HU228496B1 - Non-pathogenic mutant e.coli strains, process for production and use thereof - Google Patents

Description

The present invention relates to a novel non-pathogen. £. coli mutants which are particularly suitable for the preparation of vaccines against colibacillosis in young rabbits.

The invention also relates to the use of the mutants of the invention in the preparation of vaccines. vaccines containing mutants of the invention.

The major cause of diarrhea in young # weaned rabbits is colibacillosis. 'This disease is an important cause of death in breeding units and research has been done to produce an effective vaccine. The main vaccination strategies developed to date utilize non-pathogenic or mildly pathogenic inactivated homologous strains or live heterologous strains which induce a local immune response (induction of phylloopolyaccharide A) and / or an ecological barrier effect.

Docket; 87220-3568 / WM

So far, however, no strategy has resulted in a completely harmless vaccine that offers complete protection. Thus, research has continued to identify the mechanisms responsible for the pathogenesis of some E. coli strains.

In humans, enteropathogenic E. coli strains (i.e., .EP.EC strains), which are responsible for diarrhea in infants, are capable of inducing specific cephalic lesions called "a11achment-effacement 1esiοn", i.e., A / E. .

So fusion-deletion.i femotype, which correlates with a HeLa or Hep-2. cell line epithelial cells, defined by the FÁS (fluorescent actin staining) assay, in human EPEC strains. In this assay, cells locally adhering to the cells lead to polymerization of actin aggregation at adhesion centers, which is a fluorochrome phosphor (Knutton et al., Infect. Immun. 57, 1290 (1989)). The major determinants of this association deletion phenotype are encoded by a locus named LEE (enterocyte deletion locus) [McDaniel et al., Proc. Natl. Sci., 92, 1664 (1995)).

It has been observed that E. coli strains responsible for diarrhea in juvenile rabbits are able to induce a lesion similar to the junctional-eruption laser caused by EPEC strains in their flesh (Licoís et al., Infect. Immun., 59, 3796 (1991)). Moon et al., Infect. Iwun. 41, 1340 (1983); Peeters et al., Vet. Pathol., 22, 54 (1.985);

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Takeuchí et al., Infect. Imnn., 19, 686 (1978)], Eteket

ο; risztté strains one es authors [..Robins-Srowne mtsai, Infect . Immun., 62 3336 <19 9 4); Rofoi ns-Browne m t s a i, 1 n f e c t . Immune <, 62 1584, 1994] RE ' tribes to call (" nyűi ί · ρκ · <»í 4- »i. {Λ

The homologue of the 35 kpb chromosomal locus, originally of human origin. EP.SC strain was detected in the prototype, i.e., E2348 / 69 was found. [Karao'lis et al., In Prolongings: First International Conference on Mechanisms in Enteric Diseases *, Rapid City, 5 .D. (1995); McDaniel et al., Proc. Natl. Acad. Sci, 92, 1664, (1.9.95)] in the reference REREC strain, termed RDEC-1 (O15.H-serovar), originally described by Cantey and Blake (Infect. Dia ,, 135, 454). , 1977].

It was called an intimate., A 96-100 kDa outer memhene protein, named eaeA in the LEE lotus of the human EPEC strain. gene (Jerse et al., 1991, Infect. Immun., 59, 4302 (1991)). Donnenberg et al. (J. Clin. Invest., 92, 1412 <1993). The role of the eaeA gene in pathogenicity E2348 / 69 strains were tested in 11 volunteers given orally doses of 2.10-4 CFU. Eleven other volunteers were given the same dose of the isogenic eaeA mutant. The results demonstrate the role of the eaeA gene in virulence, indirectly, the correlation between A / Lesions, the FAS-positive phenotype, and the LEE-locus (not known at the time), as well as viru »e * *

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«Α *** · i years ago, at that time, it was thought / that the eaeA gene alone plays a role in the development of a.z A / E ~ lesion and the appearance of the FAS-positive phenotype. however, the pathogenization of the &quot; mutant residue shows that other virulene.tags are involved in addition to the eaeA gene.

In addition, the eaeA gene is also present on mem-entercathogens in coli strains [Cantey and Moseley, Infec. Immun., 59, 3924 (1991); Leory et al., J. Med. Microbio. 1, 40, 90 (9 9 3;].

The eatiA gene that smokes intixaine is an analogy to various rabbits, F. coli strains [Ágin and Wölí, In: Proceedings of the First International Rushmo Conference on the Mechanism of the Fathogenesis of Enteric Diseases ”, Rapid City, S.D., 43. (1995); Leroy et al., A. bed. bicrobiol., 40, 90 (19939; Pohl et al., Infect. Immun., 61, 2203 (1993)).

epidemiological studies in hyugst-bopa revealed that strains responsible for diarrhea during weaning of rabbits belong to a number of serum groups, the most common of which is 0103 {Blanco et al. Vet. Microibiol., 38, 193 (1994)?

CanguiIhem and Milon, J. Clin. Microbiol., 27, 743 (1989)].

The 0103 strains (which, moreover, have the distinctive property of not fermenting rhamnose), are highly pathogens in the summer following experimental inoculation by digestion and cause an attachment-deletion lesion [lioois et al., Infect. Immun., 59, 3796 (1991); Fohl and Latsai, Infect. Immun., - 61, 2203 «« «« «(1993); Bobins-Broune ss mtsal, Infect. Immun., 62, 3329 (1994); Robins-Browne ss mtsal # Infect. Immun., 62, 1584 (1994)], which are similar in appearance to those produced by human EPEC strains. This is the criterion for the generation of the lesion which, together with the fact that the chromosomal DNA of these strains hybridizes to the human EPEC reference strain. probes specific for the eaeA ~ gene [Leroy et al., u. Med. Microbiol., 40, 90 (1993); Pohl et al., Infect. Immun. # 61, 2203 (1993)] # as in RDSC-1 strain # may allow the assumption # that these strains resemble EPEC (or RSBEC) strains. However, in strain 0103, no homo aerial with any other gene of the LEE locus is detected besides the eaea gene.

E. coli 0103 strains produce a specific adhesive, termed AE / R2, which enables bacteria to adhere to rabbit enterocytes and cells of the phaLa and Hep2-2 cell line [Miion et al. , Infect. Immun. 58: 2690 (1990)).

This adhesin has been shown to contribute to the pathogenicity of strain 0103. in young rabbits. This case was demonstrated by comparing the pathogenicity of a representative strain of serum group 0103 (called strain B10) with a mutant of this strain that lost its adhesion property due to the insertion of the transposon Tn5 :: phoA into the AB / R2 adhesin coding operative. Mtsal, Vet, Microfoiol. # 50, 105 (1996)]. It has been suggested that these mutants be used as vaccines [Chalareng et al., Communicat. aux. γτ · ¹ ^ © journées de la recherche cnnicole (Proceedings of the 6th Open Rabbit Research Meeting? La Rochelle, December 6 and 7, 1994, INRA-ITAVI-ASCF Publ.}.

In order to clarify the extent of similarity-

two are coli 01.03 and tribes between vi zsgáltuk 0103 rev this is because the blood. that FASHION non like .am also; ocsolatban locus (McDaniel 1 and others, 1664 (1995) «J *

consists of EP.EC strains LEEProc. dorsal, Acad, Sci., 92,

In fact, we found that the BIO strain reacted negatively to the FÁS test, while the RDEC-1 REPEC strain and the human EPEC strain gave a positive reaction under the same experimental conditions,

On the other hand, however, it was surprisingly found that the BIO strain produced a new cytopathic effect (CPE) on epithelial cells of the BeLa cell line, which was manifested in the reorganization of the cytoskeleton and focal adhesion plaques. As with the FÁS type response, this CPE Is also correlated with the intense polymerization of cellular actin. However, in contrast to what is observed with the PÁS type response, the polymerized actin does not become localized at the bacterial contact points, but rather in the form of cables that extend from one end of the cell to the other. Such modifications of the cytoskeleton strongly and inversely increase the time. over.

In addition, the formation of actin cables is accompanied by a gradual increase in vinculin levels, a phenomenon which is not observed in the FÁS type response (Finlay et al., Infect, Immun., SO, 2541 (19-92)). In control cells, vinculin is present in the form of point masses which appear on the contours of the cells, whereas in cells exposed to BIO, vinculin is distributed throughout the cell surface, converting from a point distribution to an almost fibrous distribution reminiscent of actin cables. Such a change in the cytoskeleton. it is accompanied by a cessation of cell division and a certain increase in cell size. While the. cytostasis lasts for a long time, leading to cell death at the end of a 5-6 day period.

Examination of other strains showed that this CBE was expressed (expressed) in all rhamnose-negative 0103 strains as well as in RDEC-1. Similarly, this stone was observed in the human clinical EPEC flavor. In contrast, the human reference strain, S2348 / 69, did not show any detectable CPE under the same conditions.

The change in cyt-o-skeleton characteristic of this CPE is reminiscent of that induced by B. coli toxins CNF and CNE2 in Föswaid et al., Proc. Natl. Acad. Salts. USA, 91, 3811 (1994) 1. However, we did not find any genes in Slo that were homologous to coli CNF1 and

CNF2> In addition, while the activity of CN'Fs could be detected in lysates of cultures of CNF-efc producing strains, no activity could be detected in the messages of BIO cultures. However, it is possible that the mechanism leading to CPE induced by BIO is similar to that of CNF toxin, the latter mechanism of action associated with Rho activation, which is a small G-propcene involved in the regulation of the cytoskeleton and focal adhesion centers {Elínn and Bridley, J. Cell. Sci., 1.9, 113, 3 (1996).

In parallel, the chromosomal DbS of several 0103 strains (including prototype 810) were hybridized to 35 kb

Four samples from the LES vi.ru.lenc.ia unit were compared and the proteins secreted (selected) by strain 0103 and strain 2348/69 were analyzed by their respective electrophoretic profiles as well as by cross-immuno-precision. Strain 0103 demonstrated the presence of a locus homologous to the LSE virulence unit of 83348/69, although it exhibits some structural differences compared to the latter (which were detected by polymorphism of DNA restriction sites and less by electrophoretic migration of selected proteins). That is, the genus in the EPEC strains, which is analogous to LEE, will be hereinafter referred to as the "LEB ~ locus".

It was also observed that this CDS correlated with the in vivo pathogenicity of the 0103 strains.

Starting with E. coli O103, strains carrying mutations of this LEE locus were constructed and it was observed that a strain that is inactivated by one of its LEE locus genes no longer induces CPE and loses its pathogenicity, retains the AF / R2 adhesin and is able to colonize the gut. so the LEE locus of strain 0103 seems to be a genetic deχ * ♦ * ♦ *

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Contains terminology responsible for CPE and pathogenicity.

Finally, the immunogenic properties of these LES-locus mutants and its properties have been investigated. We found that these mutants confer a very effective protection against the pathogenic parent strain.

The present invention relates to a method for producing a mutant of E. coli, a pathogen of E. coli, from a pathogen mutant of E. coli which is capable of inducing cytopathogenic effects in epithelial cells of Hera, which results in the formation of polymerized actin cables extending from one end to another. , in part due to the increase in vincinline, characterized in that the pathogenic strain of E. coli is mutagenic and that mutants that are no longer capable of inducing cytopathic effects are selected.

The 310 strains, deposited on January 15, 1997, under the number 1-1807 'of the French National Collection of Microorganism Cultures, 28, Rue du Docteur Roux, 75724 PARIS CSDEX 15 , by way of example only, those pathogenic strains of E. coli which are capable of inducing the aforementioned cytopathic activity and which can therefore be used as starting materials for the preparation of the present invention. .

The invention also encompasses non-pathogenic mutant strains of Et coli obtainable by said method. These strains carry at least one fi * * · <Φ »Χ mutation that results in inactivation of at least one gene of the s LEE locus.

In a preferred embodiment of the present invention, the mutant strain is a .3. coli strain which is pathogenic to saliva and belongs to serum group 0103.

In another preferred embodiment of the present invention, the mutant strain carries at least one mutation that results in inactivation of at least one sep gene of the LHE locus.

Preferably, the mutant strain of the invention carries at least one mutation that results in inactivation of at least one of the LEE locus esph or espB gene signal.

A strain according to the invention, designated B1 / C & l, was deposited on January 15, 1997, under number 1-1808 with Coliection. Natiouale de Cultures de Microorganismes (CKMC) / French National Collection of Microorganism Cultures], 28, Rue du Docteur B.cux, 75724

PARIS CEDSX 15.

The present invention relates to vaccines, in particular vaccines which can be administered orally, containing at least one strain of the mutant of the invention.

Preferably, said mutant strain has a functional AF / R2 adhesin and therefore retains its ability to colonize the intestine.

For example, vaccines of the invention containing the strain B10 / CA1 can be administered orally and protect young weaned saliva from infection with pathogen E. coli strains belonging to serum group 0103.

The present invention will be better understood by reference to the following description, which provides non-exclusive examples of mutant strains. and the use of these strains as vaccines. demonstration of the ci topa ti effect created by coli 0183 strains on Béla sheikh ski tours

The bacterial strains used in this series of experiments are as follows: ·

enteropathogenic BIO and RDSC-1 coli reference strains for rabbits;

strain ΒΙδ. / 1'βΕΙ, a mutant of BIO obtained by TnphoA mutagenesis, which no longer produces the AF / R2 adhesin [Pillien et al., Vet. Microlbiol. 50 (1996) 1;

- 13 &. coli strain isolated from various cases of rabbit diarrhea and previously examined for experimental pathogenicity and for certain virulence characteristics such as AF / R2 and eaeA (Canguilhem and Miion, J. Clin. Microlb.iol., 27, 743 (1939); Leroy et al., Med. Microbiol. 40, 90 (1993) 1;

and FÁS as a control. positive human strains:

- human strain E234S / 6.9 EPBC;

two human EPEC strains labeled with CFU201 and CF517 (courtesy of Christian Foresfier, Department of Pharmacy, Clermont-Male); these strains were obtained from childhood diarrhea; they hybridize with eaeA specific and & fp ~ specific probes and respond positively to the FÁS test.

- Interaction test on Haha cells

Thus, the day before the interaction assay, HeLa cells are • germinated on split microplates {Lab-Tek, Miles Scientificj, or cell culture titration plates (NUNCj at 4 µg / ml in Eagle's Minimum Essential Medium · containing Earle salts (BMEM, G1BC0; and contains 10% fetal calf serum {FCS, GIBCO3 and 50 pg gentamicin per ml (GIBCO). On the day before the test, bacteria were cultured in "PEKASSAY broth {DIFCO}. After 24 hours incubation at 37 ° C, 5¾ In the presence of CGy, cells were washed with Earle saline buffer (EBSS, G1BC0) and mixed with bacteria in the same BMEK containing 25 mM HEFE3 buffer (GISCOI containing 1% D-mannose (SIGMA) and 5t FCS (calf embryo serum)). After incubation for 4 hours at 37 ° 0 ~ οη in the presence of 5% COg,

now ; EBSS.1, May d which Keep 10% FCS. Imaz to in order to tonkr The v. i Ö1 il > incubation time

♦ at κ * ° C) in the presence of 5% CGy, cells were washed with EBS'S and then with 1% formol in PBS. fixed for 30 minutes at room temperature.

After these different incubation times, we confirmed the morphological changes and quantified cell growth.

* Shifters at cítoazke1ebon

F actin with phalloidin linked to rhodamine. was made visible (MOLEChLAR PBBCsj under the conditions recommended by the manufacturer). Vinvirulent was detected by indirect immune £ Inesorescence using murine anti-vinvulin monoclonal antibody (V2N-11-5 clone, Sigma) at 1/100 dilution with using goat anti-mouse antibody (IMMUNOTECH 0819) The microplates were examined with an ISICA DMR fluorescence microscope (40x guideline).

The results are shown in Figures 1 and 2.

Figure 1 shows the morphological change in the cytoskeleton of HeLa cells in the FÁS assay after exposure to E. coli strain B (A and 3) and £ 2348/69. (C and D) followed by 4 hours incubation.

Staining for actin F was achieved using phalloidin / rhodamine. The same microscopic area was shown for each strain in fascial contrast (A and C) and under fluorescence conditions (B and D) at 4x × 1, line size 20 µm.

φ ΦΦ **

ΦΦ * * y 4 Κ «φ« Φ * φ Φ * Φ Φ * «

Α Figure 2 shows changes in the cytoskeleton and focal adhesion plaques produced in HeLa cells after exposure to BIO strain E, coli · 24 hours (8 and E) and 48 hours (C and F) for interaction or the mutant strain 810 / CA.l (the preparation of which is described below / in Example 2; 24 hours after the interaction (A and D)).

Left (A, S, and C): staining of as-F actin with pha11loin din / rhodaminna1 Right (D, E, and F: staining of vulin vinyl by indirect immunofluorescence using anti-vin.vul.in antibodies,

The line size is 20 μη.

* Quantification of cell growth rate quantitatively

Methylene blue is a specific method for staining cellular proteins, which allows us to examine the morphology of the cells on the one hand and to measure the total protein spectrum of the cell lawns, on the other. Cellular proteins were stained with 1% methylene blue in 0.0 M borate buffer according to the method described by tilson [Animated cell cuiture: A practical approach # R.I. Frishney (Eds.) IRL Press Oxford (199.2)] The residual protein of cell cultures was quantitated on the day of interaction and 72 hours later by extraction of the dye with 8.14 HCl and automatic ELISA plate reader (model M 5800/7000, Dynatech) measure optical density at 630 nm15

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The results are shown in Figures 3 and 4.

A. Figure 3 illustrates the morphology of HeLa cells four days after the interaction for both the BIO strain

CB} · CA for both B10 / CA1 mutant). The staining was done with methylene blue. The line length is 20 gm ...

Figure 4 shows the effect of bacterial inoculum finoculum dose on growth of He la cells after interaction with BIO (0), Blo / CAl <0} and ΒΙΟΖΙβΕΙ »£„ coli.

In the abscissa, the multiplicity of infection is the number of CKJs per cell at the start of the interaction test;

cell ordinate coefficient of cell growth - OD (Optical Density) 72 hours after interaction / OD on day of interaction.

Each point is the average of four samples. The standard deviation of the cell growth coefficient is in all cases less than

0.2.

Conclusion

As shown in Figure 1, the Blö strain does not. does not induce any FÁS type response, ie it does not lead to any significant thickening of the polymerized actin at the point of contact between bacteria and cells, and this RAS-type response occurs despite strong adhesion and in contrast to the results obtained with strain B234S / 69. , which elicits a strongly positive FÁS response under the same conditions.

ψ - * «Φ ♦ <·

A Po2it.lv FAS-type response was observed even in the case of RDEC-1 strain, despite very low adhesion, and in human CF11201 and CF517 strains.

A. aas exams £. Among the coli strains, four strains of the raianznegegative 0103 phenotype did not elicit a FÁS type response.

Still, cells exposed to the BIO strain do. show a profound change in the cytoskeleton that can be detected upon completion of the interaction (Figure 1) and increase in size over time (Figures 2B, 2C, 2S and 2F). These changes, designated herein as CPE, are they are expressed in the formation of a large number of actin cables, which are arranged in bundles across the cell and are often parallel (Figures 2B and 2C). Along with the strengthening of actin stone bundles, a gradual increase is observed in the vi.nvu.tin formations, whose foci are also associated with adhesion plaques. The chair is the formations that are usually located on the perimeter and on the periphery of cells previously exposed to the strain <RTI ID = 0.0> 2348/09, </RTI> after incubation with the BIO strain. From 48 to 72 hours, a general and pseudofibrillar distribution is observed. (Figures 2E and 2F).

The size of the cells exposed to the BIO strain is appreciably increased with time, i.e., about 2.5-fold after 4 days of incubation, consistent with the diameter of the nucleus (Figure 3); in parallel, cell growth was blocked, as was the case with residual (residual) cell cultures.

♦ * X *

Jf ♦ is determined by protein m definition X (Figure 4). The cells, only the incubation.

In addition, calculated as i, i col ony- .forrni ng

50% of the day shown to begin to die.

it was estimated that at the start of the test, the cell BIO is a graft of 50-100 colony forming units (CFU) at least

CPE.

Although the BLO / 16 IS XIVIL; <3.11 £.> no strain at all form fibrillar adhesin (Pilil et al., Vet. Microbiol. SO, iS (1996)), ' ^! · brings the same CPE created as a parent <· BIO job RZS; as however, it is shown in Figure 4 mutat-

ya, cytopathogenicity kb, SO fold lower.

A. Among the other strains from rabbit diarrhea examined, eleven strains with the same phenotype as Blö <0103 serum / rhamnose fermentation, eaeaa positive}, produced all morphological changes defining CPE. This CPE is induced by three of the other six strains examined, as well as the reference R.DEC-1 strain. Of the human EPEC strains, E2348 / 69 did not produce any detectable CPE, whereas the two CF112O1 and CF51? strain were very positive for CPE,

A. CPE could not be reproduced using the 10-fold concentrated supernatant of the BIO interaction culture, nor the lysate (solutions) of the bacterial bodies obtained from the same culture and similarly ten-fold concentrated.

2. pe.íi'.líJ

Production, selection and genetic vice versa of CFE-negative mutants

In order to gain insight into the molecular target region of this C? E and to investigate the possible correlation between CFE and ectopic pathogenesis in rabbits, we have isolated tin from the bank of B1? Mutants that have lost their ability to induce CFE. it was obtained by randomly inserting the transposon TnSISSOjy: pho.4 (TnphoA) (Manoil and Beckwith, Froc. Natl. Aead. Sex., §2, 8129 (1935)).

TnphoA is introduced by conjugation with the suicide plasmid p.R7733 into the BIO strain of K. coli strain SH1Q (kpi: r +) (Taylor et al., J. Bacteriol., 171, 1.870 (1989)). .

The transposon 'received clones were selected in two steps:

1) The conjugation mixtures on Luxia-Bertani agar (LA medium) were first enriched in the transposed clones by transferring them to a M9 minimal medium containing kanamycin (20 pg / ml) and incubating for 48 hours at 37 ° C. strains do not grow in this medium because SM1.0 is auxotrophic (thy thr les) and BIO is sensitive to kanamycin.

2) In a second step, the enriched cultures were plated on LA medium containing kanm.ic.int (50 pg / ml) and 5-bromo-4-chloro-3-in.dol.yl phosphate (X?) (40 pg / ml). 40 pg ~ mi). Colored in blue clans then phenol red aaarra • ί replikáituk _z containing 1¾ rhamnose and LA medium containing either kanamycin (100 .mu.g / rai} or ampicillin (100 μ g / ml) contained, in order to elim.inálja Clones in which your entire pRT733 plasmid is co-integrated with BIO ge & oaaal ·

Clans that had the rhamnose negative phenotype and kanamycin resistance as well as ampicillin sensitivity were retained for further study.

The mutants thus selected were then cultured overnight in PENASSAY medium containing kanamycin (50 µg / ml) and then added to glycerol (final concentration 20¾} -20 ° C.) .96 hollow cell culture on plate (FALCOK) their GPS-triggering ability on HeLa cells was determined using the methods described in Example 1 above.

Of the nearly 2,700 clones examined, 7 were independent clones (i.e., clones isolated from independent conjugation mixtures) which were CPE negative and did not integrate the entire suicide plasmid.

Figure 2 (A and B1 illustrates that one of these mutants (B10 / CA1}) has lost CPE. The figure shows that cells exposed to this mutant show no change in the oocyte skeleton, which is wild type B10). strain-induced CP'E trait.

Figure 3A also shows that the B1Q / ÁC1 mutant does not cause any morphological change induced by strain B10. Figure 4 shows that the cytopathogenicity is almost zero, the mutant a

Harbor, NY ( 1983)) s The C? E ~ negative definition to your goal Boehringer) help < 3.4 kpb Hindi .11 (DIG-UTP). ) irt a ε

Genetic characterization of C.PE-negative mutants

Unless otherwise noted, the techniques used are described in Sambrook et al. (Holecular Cloning: The Laboratory Manual # Co'ld Spring: 1989)].

-in order to count the number of units inserted in negative mutants using a commercial kit (DIG DNA). .1 opposite.

THE ? we removed two of the pre-selected clones # because they contained double inserts.

The insertion in the remaining 5 mutants can be localized as follows:

We also observed # tips on the BIO chromosome tower with DNA for the LSE focus of strain E2348 / 69 described by McDaniel et al., (1995) Proc. Natl. Acad. Sci., 92, 1664. (marked A, C and D in Figure 5)

hybridize it. # as soon as the Fig. 5a presents. This is the hybrid! cié and you bb like 3 5 kh region t happens (as in Fig at ja), as U2ly f that's why ' it seems that E2 3 4.8 / loci analog Toss' forms a uset, a restriction polymorph! it is in us tin sign self Ancient Küiönbséoe] they are.

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The probes were prepared by labeling fragments A, B, c and D with digoxin-DIP, digesting the complete DNA with various restriction enzymes, and then carrying out the Southern biot on the CPE-negative mutants with these probes. profiles were compared to parenting in autumn. BIO strain and S2348 / 63. V

Apply these probes to it. concludes that the five mutants have inserted the TnphoA insert into the region corresponding to the LEE-horse. Figure 5b shows the position of the TnpboA insert in this mutant with a circled arrow.

In three of these mutants, {BIO / CA1, BIO / S2, and B10 / PA12}, the insert was located in the 8 kB ScoRI fragment which hybridized to the 3 probes, i.e., the secretory {.sub.2} genes (sepas - sepD} homologous genes

ebony <. THE BIO / HL2 ridizáiő regions sban it ergo and

kind of elk example

Protein Comparison of the protein profiles of TAS responding strains, CPB inducing strains and CPE ~ o-s.gat.i.v mutants

In order to identify any potential bacterial protein selected that may be associated with CPE, we analyzed the electrophoretic migration profile of proteins in the culture supernatant at <RTIgt; £ 2348/6: 9 "*" * · * "4" "Λ *

Λ> ** ♦

* * for human S.PEC strain, BIO ', RDEG-1 and S10 / 16E1 strains, and CFE-negative BIO mutants.

The bacteria were cultured under the conditions of the interaction test described in Example 1 above, but in the absence of cells, the initial inoculum was approximately 10 ⁷ CFU / µl. After incubation for 2 hours, the bacteria were centrifuged (4000 g, 15 min, at 20 ° C and 1 mL of methionine-free EMEM (No. 31900-020, GlüCO; suspended in containing 5% additional dialyzed FC5) for 20 minutes. after incubation at 37 ° C to deplete the endogenous methionine pool, 100 µCi / ml of methionine labeled with - 5 S. After another 1 hour incubation, the bacteria were removed by two successive eemrifugations (12000 g for 10 min). followed by centrifugation in the supernatant, the proteins were precipitated with 40,000 polyethylene glycol (PSG, 200 mg / ml, MERCK). μß was suspended in electrophoresis loading buffer and the proteins were separated by electrophoretic migration on a 12% polyacrylic Hantid gel in the presence of SDS (SDS-PAGE). and contacted with a β-msx (AMEASBAM) film at room temperature for 6 days. The film is developed as usual.

The results are shown in Figure 6A.

Three major proteins with molecular weights of 39, 37 and 25 kDa and a minor band at 40 kDa, respectively, were identified in the Blö parent strain (lane 3).

X * · Φ ΦΦΦ * X * φ · * '♦

While the profiles of rabbit RBBC-1 and B10 / 16EI mutants are identical to those of Blö, the human SPEC reference strain, £ 2348769 (lane 6), shows four bands with relatively similar molecular weights of NO, 39, 36, and 26 kDa) and one. another larger band approx. 29 kDa, which has no equivalent in the BIO profile (Figure 6A). None of these stripes. appears in the CPE-negative B10 / CA1 mutant. (Lane 4). Lane 2 contains molecular weight markers.

These proteins are immunological. and polyclonal rabbit serum produced in serine with PEG precipitated BIO supernatant proteins.

Immunoprecipitation with antiserum to the supernatant proteins was performed on the culture supernatant after being labeled with - S as described above. Immunoprecipitation was performed using standard methods [Sambrook et al., Moecuiar Cloning: Laboratory Manuai, Cold Spring Barbor, b.Y. (1989) J.

The results are shown in Figures 6B and 7.

Footnote to Figure 6B: Lane 1: BIO with preimmune serum; 2: BIO; 3: BiO / CA1; 4: B1Q16E1; 5: molecular weight markers; 6. BDSC-1; 7: £ 2348/69.

Figure 6B shows that the antiserum has BIO {3.

S £ V · and RBSu-l (lane 6) with its main protein (39, uh? 5 25 kDa) as well as £ 2348/69 gy pro te injured1 (39, 36, 29 26 : Da) precipitate. While BIO is three main prof

ine is present in the AF / R2-negative mutant (B.1.0 / 16E1 # lane 4), does not appear in the CPE-negative mutant (Blu / µAI lane 3), but it should be noted that serum also has a degree of ability to bind the BIO with the 40 kDa minority protein

immunpr ec í pi ti t-u? to form salt. These ing, that Blö supernatant pro teinj of and as E2 348 / 69 (still if this:

It is also perceptibly different) and that the formation of these proteins in the CPEnegative mutant changes in the supernatant.

To decide that this disappearance of protein in the supernatant a. we compared the ismmnprecipitating profile of the supernatant proteins of the BIO strain and CPE-negative mutants (CA1, FA12, E2, A9, and H12) to lysates of bacteria from the same culture, or due to a defect in bacterial secretion with an iffimmune prediction profile.

The lysates were obtained by three freeze-thaw cycles (-20 ° C to 20 ° C) followed by centrifugation (12000 g, 10 minutes) and the supernatant of the lysate was recovered. Immunoprecipitation was performed after labeling - ^ ^-ηοΙ under the same conditions as used for the supernatant proteins. However, before this was done, an additive from the B. coli HBlöl laboratory strain was used to deplete the antiserum to eliminate non-specific reactions.

supernatant proteins

7B (bacterial

The results are shown in Figure 7A (immunoprecipitation profile of lysmunoprecipes) and lysates.

Footnote a 7A Figure 1: bar OK 1: BIO; 2: pier and markers; 3; CA .1; 4: FAI2; 5: E2; 0 : AB; 7: H12, Figure 7A shows it mt watering that dig hlG- razor blade and eluted compared in

bypass proteins are significantly reduced in all mutants. This decrease seems to be even more pronounced in three mutants, namely CA1, Ε-2 and A9-foen. Immunoprecipitation shows a band of approx. 110 kDa, the intensity of which is more or less the same in all strains and is therefore unaffected by mutations.

Note to Figure 7S; bands; 1; molecular weight markers, * 2; BIO; 3: CA1; 4; FAI2; 5: 2B7 6: A9; 7: H12.

Figure 7B shows that the major proteins described above are present in more or less equal amounts in the BIO lysates and in the. mutant lysates except for the CA1 mutant, which appears to be particularly poor in the 25 kDa protein.

Example 4

Loss of pathogenicity in a CFE-negative mutant of BIO

To study whether the loss of CPE has an effect on virulence, we compared the rabbit pathogenicity of the BIO strain for oral administration with one of the mutants, in this case the B10 / CA1 strain.

Pathogenicity was examined on 35-day-old young saliva from New Zealand white culture (INRA strain).

φ φφ ί *** * »* *« · ** ♦ * * φ * * φ »♦ * * * ί * _Λ φ Κ-.φ Λ Φ» # • φφφνφ which. At 28 days of age, they were separated by the method described by Pillien et al. [Vet. Microbiol. 50, 105 (1996-1991) 51 livers were divided into three groups which were homogeneous in weight and litter and then inoculated orally with the 2.1 <P CFU unit of the test strains, namely: the Blö parent strain (18 animals), with the CFB-negative B1 / CA1 mutant (13 animals) and a non-pathogenic KI2 laboratory strain BM21 (1.5 animals). Diarrhea and death, as well as weight, were scored and B. coli strains were counted and classified as it is described by Billien et al. (1996, ed.).

Oral inoculation with the BIO parent strain caused weight loss in all 18 vaccinated animals, including 17 cases of diarrhea and 16 deaths. The deaths occurred between the third and the twelfth day after vaccination. for comparison, animals vaccinated with the same amount of B1 / CA1 mutant, similar to those vaccinated with B. coli K2 strain, showed no weight loss or clinical signs. The weight curves of the latter two groups were practically identical.

Figure 8A shows den group in the experiment: BIO (two living / 18 animals Δ; K12 (control; 15 surviving / 15 animals) - 0; B10 / CA1 (18 surviving / 18 animals) - □.

Figure 88 shows the population of coli hacillusofc at different times after inoculation in the faeces of animals that were vaccinated with the K2 control strain, the Blö strain and the Blö / CAl strain:

average weight of surviving animals min · □: Collbaciilus flora identified as Blö / 'CAl

Q: Colibaoillus identified as a BIO strain

f. 1 hour.

Significant colonization (proliferation) of S. coli was observed in rabbits vaccinated with the parental SIO strain, and colonization reached a level of .10 CFU / g in survivors more than two weeks after inoculation. All identified colibacillus flora contained BIO strains.

This total E. coli population was ICp-10x-fold greater than that seen in the rabbits in the control group, which were inoculated with the Klz strain of E. coli, which was not. have also been detected in appropriate animals.

In lymphocytes vaccinated with the S1 / CA1 mutant, the latter colonized the intestines at high levels, after which the E, coli population stabilized at about 1.5 µg / CFU / g for at least two weeks. During this time, '? 0 ~ 0%' of the total E. coli population was B10 / CA1.

She. oe 3da

Relationship of protection conferred by a CBE-negative strain to infection with parental strain B20

The effects of inoculation of the pathogenic BIO strain with a dose of 10 <RTIgt; CFU </RTI> on a daily basis were compared with 2, 13 of 35 subjects, one of which had a week φ * *.

X Φ * * «φ · ♦ ·>« * * φ

Λ * * * Previously orally administered mutant strain B18 / C & 1 (2xl1) · '

CFU!

Thirty-six young bunnies (species and breeding conditions identical to those described in Example 4 above) were divided into two groups which were homogeneous in weight and litter. At 30 days of age, animals in one of the groups were dosed with 2 x 10 4 CFU B1Q / CA1 mutants orally in 2 ml PBS. Seven days later, a dose of 2x10 4 CFU (corresponding to a lethal dose of about 150 S) was added to both groups, orally, from the parent pathogenic parent BIO strain. Diarrhea »and deaths as well as weight were recorded and £. coli strains were counted and sorted as described in Example 4 above.

Throughout the observation period, no clinical symptoms or weight loss were observed in young rabbits previously dosed orally.

Strain B10 / CA1.

On the other hand, out of 18 saliva, 11 of those unvaccinated with BIO / CA1 showed weight loss, while 9 of the nynlak showed diarrhea and 7 of 18 died,

The intestinal colonization assay showed that the B10 / C & 1 strain effectively colonized the intestines when the pathogenic BIO parental strain was administered; then strain B10 / CA1 at ca. 10 'CFU / g remained stable for two weeks, thereby preventing B10 from being detected at a detectable level.

* * * * ♦ x «i

Parallel ELISA was used to measure local anti-0103 LES XgA antibodies in stool. Figure 9 shows the development of IgA antibodies in stool after oral inoculation with CPE-nsgativ 310 / CA1 strain (day 0) followed by virulent strain 810. after vaccination (day 7) (0) or only after vaccination with virulent strain 810 on day 7. It can be seen that the antibody response is significantly increased approximately 15 days after oral vaccination with strain B10 / CA1.

She. example

Isolation of a LEE mutant to look at a site specific mu tag

The right end of the LES focus of strain B10 was cloned and sequenced. The sequence of the sequenced antigen is shown diagrammatically in Figure 10 and the resulting sequence is depicted in SEQ ID NO: 1. In addition, we examined the general organization of the LEE1 focus in the pathogen 0103 strains and confirmed that it was identical in all of these strains.

The LES locus mutants of strain 0103 were generated

Starting with the E22-labeled mutant strain, While this pathogenic strain is analogous to the S10 strain, it is sensitive to streptomycin {as well as all Graa-nagativ spectra examined tibo t i Rumra}.

These mutants were produced by the ai exchange method.

For each selected locus, the allele of {eaeA, espA, espB, and espDi in the wild-type strain E22 was replaced.

S22

Λ ¹ ♦ * ί

fc- «« •• Οί'ί * - +

Φ »•« · «· ♦♦»

* φ ♦

φ * »« «with an allele # contained in an expression cassette [-Galan et al. # 174 # 4338 (.1992) 1] contained an expression gene for kanamycin (aph.T gene: amlnoglycoside 3'-phosphotransfer) under the control of a strong pr omo te.r # without a transcription terminator. This cassette #, flanked by the genes to be mutated #, carrying a suicide plasmid [Kaniga et al. # Gene # 109, 137 (.199.1)] is introduced into a wild-type strain and selected into recombinant bacteria. the mutated allele replaced it.

The eaea and espoo genes were inserted into the natural Eco-RV or cassette. by inactivation of BglII at the restriction site. For espB and espA, de. Novo is a SgJJT (GACATCj site was created for site-specific mutagenesis.1 (using PCR degenerate oligonucleotides and restriction plasmid selection).

Proteins produced by the resulting mutants were analyzed by immuno-b-Lotto for EaeA and for isaolypi recipients. for EspA, EspB and S-s-pD. This analysis confirmed that these mutants differed only in. wild-type strain that did not produce the protein # whose gene was inactivated by the mutation.

The resulting mutants were:

- EzzAespA # which no longer synthesizes EspA protein, ~ E22AespB # which no longer synthesizes EspB protein.

- E22AespG #, which no longer synthesizes EspB protein.

- S22AeaeA #, which no longer synthesizes EaeA and therefore does not incorporate it into its outer membrane.

# *. <ν *. * * * '* ·.

«8 <fr ♦ '* ♦ * ♦ *« - «1 \ * ♦ φ 9 * ♦» «* <· * **' '*

Synthesis of other proteins remains essentially normal in each mutant. For example, the E'2'2αespA mutant no longer produces SspA, but normally secretes EspB and EspD just as it normally synthesizes and integrates EaeA into its outer membrane. Thus, each of these mutants appears to carry a strict non-polar ("saris, non-polar") mutation that affects only the target gene of the mutation.

Figure 10 shows the right end of the LEE locus of strain 0103 and the position of the various genes, as well as the location of cassette insertion in each of the 4 mutants obtained.

These mutants were subjected to in vitro assays to determine their ability to induce CPE in HeLa cells as described in Example 1 above.

The pathogenicity of the esp-gen mutants was also checked in isolated rabbits by administering 2x10 '' CFb mutants per group of 35-day-old rabbits orally using the same protocol as described in Example 4 above.

* ♦ »« «*** * ♦ * ♦) ί» «. ** * * »*. *. ♦ * X *« A X X * κ «« «« ««> »» «*»

- 32 ~

The results obtained are summarized in Table 1

1, Table

Tribe  CPE / Keka vivo patogentíás ³ süiyveszte- ness diarrhea mortality £ 22 * 4-F + 23/34 26/34 25/34 E22ÁespA 0/16 0/16 0/16 £ 22AespB - 0/16 0/16 ' 0/16 E22Áesp £> - 3/18 2/18 2/18 E22ÁeaeA • S-4-4- NT NT NT

a: Number of animals showing clinical signs / number of vaccinated animals *: wild typ.su strains

DK: not exams,

These results indicate that Esp protein synthesis mutants already express the cytopathogenic effect on Heha cells as opposed to the eaeA mutant. As a result, two .Esp protein mutants (ÁespA and ÁespS) have completely lost their pathogenicity and the pathogenicity of the ÁespD mutant is much lower than that of the wild-type strain.

SZEKVENCIÁLISΤΑ

1.2 1. SEQ ID NO: 1 SEQUENCE CHARACTERISTICS:

LENGTH: .12620 base pair TYPE: nucleotide

THREE FIBER: Double

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

FEATURES:

NAME / KEY: cds

E LHELYE ZKEUÉ S: 1768, .. 4587

OTHER INFORMATION: / produk - eaeA FEATURES:

NAME / KEY: cds

LOCATION: 7278.. .7856

OTHER INFORMATION: / product = ^w espA ^{! 5} FEATURES:

NAME / KEY: cds LOCATION: 7869 ... 3011 OTHER INFORMATION: / product = ^M espD * FEATURES :.

NAME / KEY: cds

LOCATION: 9032 ... 937 6

OTHER INFORMATION: Zprodukt - ”espB”

DESCRIPTION OF SEQ ID NO: 1 SEQ.

OATATWCCO AACCCCGTCA CWSATMTA SmWAOTOWM T & CMAffiMC SÖ & CG & WASC CASGffiS & TCC AA4TASTGÖC Ö & OSA & A & TOmWSAT TCATACtTCA X2Ö

ACCTOACTT CMCCTCCS TÖCAÖATCCT AAACTTW3T TSTShOT SACTATTGCT 188

8CM3OTSTO & TAGGÖATÖOC TGCGACGGGS & TTGC & C & GG OTGTTGCWT SACTCCAOAÖ 24ö

CCGG & TO & CC CAATCACTAC CÖACCCTÖAT GCTGCMCAA & C & Ö & 3CTG & mOJ® 388

AAAÖATCAST TAACOWO, ASCMTSOS AACCCAGATA AŐbSAMST ΤΜΤΑΤ03ΑΤ 388

8AÖAACG0AA ATGCÓATTCC STOT $ KM CTO && 3 & TG ATSTTÖTTSC GCAAATAGCÁ 420

S & AC & ftSCTA AAÖCSOCÖGG TÖAACN38CC A®CO «CTATTGAAAG TMTTCTCW 488

SCGCAGOAA JWATOAWL KCAÖOmCT AAACSO3MC K «MW TCOTTCATCG 54 S

GQGGrmOCT ACOSTATTAS XWTGCGCKS ATTCTTOOS GGGCAATTGG TÖCCT3TOT? 888

Μ 9 »X XX * * **

* φ - 35 -: ... ¹  I ♦ ♦ ♦♦ *:> ACTGCCTCTC TTC & TCGCAA AAACCAACTS δΜΑΑΟΜ CAATt & CTAC ACGTACTGTA 888 STCG & TAATC AGCCTACGAA TAACGCACTT GCGC & CTGCA ATACTGACAC AAGTGGSCCA 728 GAAÖAGTCCC C3GCGASCAG ACTTAáTTCG AATGCCAGCC TCTCATCGA & CCTGTCTGAC 788 ACCTCCAGCA CGCTCACGGT AGAGAATCCG TACTCTGACT TCTGAATGCC CAGAAACTAT 850 TCACCTGCTC GCATTTC & GA G8AACCTATT TATGACTAW TCGCCTCAGA TCTTAATTAT 208 AGCGTCATTC AACATTTTTC A8CTAACAGC KWW GAACTTTAG? GCTAACCCCA Μ8 GSGCAAGGTA TCCAAAGT & C TTATGCTCTT CTOWa GÖSSO0S & TT GCTTTTAGGT 1823 ATCTGACTAT TAACGGGGCT GCTCGAGAOC GCMGTAAGTA CCTCCAATGC CGCACCAACG A, » ^v 1888 CCSGGACCCT CACGTTTCGT TTAAATATAT CCTTGAGTAT TTAGTCTACT TTGGGAGGGG 1148 GGGACTTTTA CTAATATTAA CTTTCTAGAG AATAAAGTTG CAGCACTGÖT AACTCTTGAA 1288 CTTCTOTAAT TATAAATTCA ATTAAGMGAA ATT & TAACT? CATCAAGATC CTACCTTTTA T2CO T7AGATAGAT TTSCACAAAA AATTCTTATT GGATCTATCT αΏΏΛ®Α AAATAGATTG 1820 worr® ctatctatga gattcattat atttcgttat ctgatgctaa ctacgaatat 12 SS ACTATGATCT & TGSCGTCTG TGCTAAATTC CCGACAGATA ATÖCTRACTT CGCTCTTGAG 1448 ATTTCTAATG CAAACTTACT GTTTGCAGAG AATCTCTGAC CATACCTGTG TTACTAATCA  > *> ·. Λ '· · ·>. The í:> CTAGCACAAT CTCWTCTTT AGCTTTACTT TTTCCSCTCT ACTATGCTAC ACCTGAAAAA aSi. , X «? Y« 5 ·? * 'Íf CTWAGAATG AAATAGAAGT CTTCSTTAAG TCAATW & AA ACCCTTATTT GGTATTACAT 2588 1558 1520 WKO »TAACATTAGA AAACÖAACAT AWAA && TAG ACTAAATTAG TTCA & SCTAT 1888 AATAAACATT ATTACTCCT © AAGATAAAAG ATTATTTATT AATATAATTT ACTATTCATT 1740 CTAACTCATT GTGGTGGACC CCATAAC ATG ATT ACT CAT GGT TTT TAT GCC Met Tie Thr Kis Gly Lhs Tyr Aia 1 5 1721

CTG ACC CGG CAC AAG'GAT AAG CTA AAA AAA. ACA TTT 'ATT ACT CT? AGT Arg T & r Arg Little Lye / His Lys Leo Lys Lys Thr Lhe.lle Met Leu Ser 18 l IS 28 ' x <X?> VV Λ «·> · <· * · \? · * · * $ -. · GCT GG? TTA GG & TCT TTT TTT TAT GTT AAC CAG AAT TCA TTT GCA AAT Ale Gly Leu Gly Leu The Fhe Tyr Vei A »» Gin As »Ser The Ale A®» 25 20 25 88 1832 1887 GGT GAA AAT TAT TTT AAA TTG AGT TCA GAT TCA AAA CTG TTA ACT CAA Gly Glu Asn Tyr Phe Lys Leu Ser Ser Asp Ser Lys Leu Leu Thr Glu 45 50 55 1535 AAT GCC GCT CAG GAT CSC CTT TTT TAT ACT TTA AAA ACA GGT GAA ACT As »Al Ale Gi» Asp Arg Leu Phe Tyr Thr Leu Lys Thr Gly Glu Thr 88 85> 70 1283

Χ «V« ** * * «ΦΧ <44

/.4 * *

GTT GCC AAT ATT TCT AAA Lys TCA Ser CAG GGT ATC AGT TTA TCG ÓTA ATT TGG 2031 vei Ale Asn.Tle 75 Ser Gin Gly 80 H e Ser Flax Ser 85 With Ile Trp TCA CTG AAT AAA CAT TTA TAC AGT TCC GAA ACC GAA ATU ATG AAG GCT 2075 Ser Lea Asn Lye SÍ® Flax Tyr Ser Ser Gin Ser Gin Met Mer Lys Ale PO PS 100 GGA CCT GGT CAG CAG ATC ATT TTG CCA GTC AAA AAA ere TCT GTT GAA 2127 Sly © Pr Gly Gin Gin He He Len Pre Len Lye Ly® Flax Grayling With Gin 105 110 22S 120 TAT AUT GCC TTA CCT GTC TTA GGT TCG GCA CCT GTT. THERE GCT GCA GGT 2175 Tyr Ser Ale Len Pro vei Flax Gly Ser Ale Pro With. With below Als Gly 12K 130 5 *?> K 135 GGT GTC GCT GGT CAT ACG AAT AAA ATS ACT AAA ATG TCC CCU GAC CCS 2223 Gly With Ale Gly Mis Thr Asn Lys heh Thr Lys Bee Ser © Pr asp below 140 $ 14 150 ACT AAA AGC AAC ACG ACC DAM GAC AAG GCT CTA AAT TAT GCG GCA CAA 2271 Thr Lye Ser Asn Thr Thr asp Asp Lys Ale Len Asn Tyr Down Ale Gin 155 140 185 CAG GCG GCG AGC CTT GGT AGC CAG CTG CAG TCG CGC TCA CT® AAC GGC 232.P Gin Ale Ale Ser Flax Gly Ser Gin Len Gin Ser Arg Ser Flax Aon Gly © 27 17S 2P0 DAM TAC GCG AAA DAM ACC GCT CTT GGT ATG GCC AGG AGC CAG GCT TCG 2387 asp Tyr Down Asp. Thr Ale Len Gly Met Ale Ser Ser Gin Ale Ser 185 © ss 185 200 TCA CAG TTG CAG GCC TGG TTA CAA CAT TAT GGA ACG GCA G & G GTT AAT 2415 Ser Gin Len Gin below Trp Len Gin Ki® Tyr Gly Thr Ale Gin With Asn

205 220 215

CTG CAG AÜT GGT AAT AAC TTT GAC SÜT AGT TCA CTG GAC TTC TTA TTA · 24S3

Len Gin Ser Üly Asn Asn Phe Asp Sly Ser Ser Len Asp Phe Asn Len

220 225 230

CCG TTC TAT GAT TCC GAA AAC ATG CTG GCA TTT GGT CAG GTC CST GCG 2511? »Phe Tyr Asp Ser Sin Asn Mer Len Ale Phe Gly. Sin v & I Gly Alá

235 24 ©. 245

CGT TAS ATT GAC TGC CSC TTT AGG GCA AAT TTA CGT GCT GGC CAG CGT 2SS9

Arg Tyr Tle Asp Ser Arg The Thr Ale Asn Len Gly Ale Gly Sin Arg

2SÖ 255 25 ©

TTT TTC GTT CCT GAA AAT ATG TTG GGC TAT AAC GtC TTC ATT GAT CAG 2507

Phe Fte Len. Pro Gin Asn Week L «u Gly Tyr Asn Vei Phe lle Asp Sin

255 272 275 28 ©

GAT TTT TCT GGT GAT AAT ACC CGT TTA GGT ATT SÜT GGC GAA TAC TGG

Asp Phe ser Sly Asp Asn Thr Arg Len Gly Π® Gly Gly Gin Tyr Trp

255 25 © 2PS

2585 * * * · »* '·· *, 5. * Χ λ r» * s

CGA SAS TAT TTC m AGT AGC GTT AAC CGC TAT TTC CGC ATG AGC GGC 2783

Arg Asp Tyr Phe Lys Ser Mud Val Asa Gly Tyr Phe Arg Mer ser Gly

388 385 Γ 3X8

TGG CAT GAG TCA TACjAAT &% 8 AAA GAT TAT GAT g | g CGC CCG GCA AAT $ 751

Trp His Glu Per Tyr Asa Lys Lys Asp Tyr Asp Óla Arg Pre Al® Asa

335 320 333

GGT TTT GAT ATT CGC TTT AAT CGC TAT TTA CCA TCA TAT CCG GCA 27 »

Gly Phe Asp XXe Arg Phe te Gly Tyr Leu Pre Ser Tyr Pre Alá he »

133 333 343 ®C GCC AAA CTG ATG TAT CAA CAG TAT TAT CGT CAT AAT GTT GOT TTG 2347

Gly Alá Lys he »Mer Tyr cl» Gla Tyr Tyr Gly Asp Asa v®X Al® Le »

343 358 355. 358

TTT AAT TCC GAT AAG TTG C&G TCG AAT CCT GGC GCÖ GGG ACC GTT CCT 2885

Phe Asn Ser Asp Lys Le »Gl® Ser Asa Pre Gly A1 & Al® Thr Vei Gly

355 373? 373 * \. ^w

GTA AAC TAC AGT COS ATT CCT CTG GTG ACG ATS CSC ATC GAT TAG CGT 2,343

Val Asa Tyr Thr Pro lle Pro Leo Vei Thr Mefc Gly Ile Asp Tyr Arg

388,335,353

CAT GGT ACG GGT AAT GAA AAT GAT CTC CTT TAC TCA ATG CAG PTC CGT 2 »1

His Gly Thr Gly Asa Gla Asa Asp Leu he »Tyr Ser Mer Gla Phe Arg

385 403 435

TAT CAG TTT GAT AAA CCG TGG TCT CAG CAA ATC GAG CCA CAC TAT GTT 3333

Tyr Gla Phe & sp Lys Pro Trp Ser Gla Gla lle Glg Pro Gla Tyr v & l

4X8 433 423

Λ

AAC GAG TTA AGA ACA TTA TCG GGC 'AGC CGT TAC GAT CTG GTT CAG CGT 3357

Asa Glu Leu Arg Thr: Les. Ser Gly Ser Arg Tyr Agg Leu V®1 Gla Arg

425 433 435 · '440' s

AAT AAC AAT ATT ATT CTG GAG TAC AAA AAG CAG GAT ATT CTT TCT CTG 313S

Asa Asa Asa He He Leu Glu Tyr Lys Ly® Gla Asp 21® Leu Ser Leu

445 453 455

AAT ATT CCG CAT GAT ATT AAT GGT ACT GAA CAC AGT ACG CAG AAG ATT 3133

Asa lle Pro Mis Asp He Asa Gly Thr Glu His Ser Thr Gla Lys He

458 455 478

CAA TTG ATC GTT AAG AGC AAA TAC GGT CTG GAT CGT ATC GTC TGG GAT 3231

Gla Leu He Val hy® Ser Lys Tyr Gly Leu Asp Arg He Val Trp Asp

475,400,485. '* ·

CAT AGC CCA TTA CGC AST CAG GGC W CAG ATT CS CAT GGC GGA AGC 3272

Asp Ser Al® Leu Arg Ser Gla Gly Gly Gla He Gla His Gly Gly Ser

428,425,588

CAA AGC GCA CAA GAC TAC CAG GCT ATT TTG CCT GCT TAT GTG CAA GCC 3327

Gla Ser Al® Gla Asp Tyr Gla A1 & He Leu Pro Alá Tyr v Gla Gly

585 510 515

520 »X« r ♦ X «* * ♦ fi ♦ X ♦ ♦ Λ

CTC AGC AAT ATT TAT AAA CTG ACC GCT CGG GCC TAT GAC CGA AAT GGT 3375 ^W Ser Asn He Tyr Lya 525 Vei Thr below Arg Alá 530 Tyr asp Arg Asn 535 Gly MT AGT TCT AAT AAT GTA C $ 3 CTC ACT ATT ACC GTT TTA CCT AAT CTG 3433 Aon Ser Ser Asn Asn With Gin Flax Thr lie Thr With Flax © Pr Asn GXy 540 545 550 EAGLE GTT GTG GAC CAG GTT CTG GTA CARPENTER GAC TTT & CT GCT DAM AAA ACA. 3471 Gin With With asp Gin With Gly vei Thr asp The THP below asp Lya Thr SSS 550 555 TCG GCT AAA GCG DAM GGC OVER THE GAA GCT ATT ACC TAT ACC GCG ACG GTT 3515 Ser below Lys below asp GXy Ile Gin Aia Ile Thr Tyr Thr below Thr With §70 575 550 W AAG MT GGT GTA GCT CAG GCT AAT GTC CCT GTA ACA TTT AGT ATT 3557 Lys Lys Dig GXy vei below Gin below Asn With prn Val Thr The Ser XLE sss 550 555 500 GTA TCT GGG AGT GCT AGT CTT GGG GCA AAT AGT GCG AGA ACG DAM CTT 3515 With Ser GXy Thr below Thr Flax GXy aXa Asn Ser below Arg Thr asp Gly SSS 510 $ 3 5X5 AAC GGT AAG GCG ACC GTA ACG GTG AAG TCG GCT GCT CTA CAG GTC 3583 aan Gly Lys below Thr vei Thr Flax Lys Ser below Thr Pro GXy Gin With 520 535 530 GTC GTG TCT GCT AAA ACC GCT GAG ATG ACT TCG CCT CTT AAT GCC AGC 37X1 vei VHL Ser below Lys Thr below Gin hhfc Thr Ser Pro Flax Asn A1 & Ser ANCESTOR 540 545 GCG GTT OVER THE TTT GTT DAM CAA ACC AAG GCC AGT ATT ACT GAG ATT AAG 3755 below With tle Phe With asp Gin Thr Lys below 5er Ile: Thr Gin Ile Lys 550 555 SALT $ GCT DAM AAA AGA AGA GCG EAGLE GCA DAM CTT TCT DAM GCG ATT ACC TAT 3307 below asp Ly® Thr Thr below Lys below asp GXy Ser below Ile Thr Tyr sss 57® 575 u / 580 ACT GTC AGA GTG ATG AAG GAG GGG GCA CCC GTA GTA DAM CAG AAA GTG 3,855 Thr With Arg With msr Lys Gin Gly below © Pr vei With asp Gin Lys With 555 550 505 AGC TTT TCT AAG DAM TXT GCG ACC CTG AAT MG ACT GAA GCA ACA ACC 3803 Thr The Ser Lys asp Phe GXy Thr Flax Asn Lys Thr Gin below Thr Thr 700 705 710 DAM CAG AAT GGT TAT GCT ACT GTA AAA TTA TCA TCG AAT ACT CCT GGC 3351 asp Gin Asn Gly Tyr below Thr With Lys Len Ser Ser Asn Thr © ar Gly 7x5 728 725 AAG GCC ATT GTT AGT GCA TODAY GTG AGT GGA GTA GC ^ ACA GAA CTT MG 3333 Lys below tle vei Ser below Lys With Ser Gly with GXy Thr Gin With Lys 730 735 700

404?

* Χ «Φ **

OCT ACT ACC OTT GAG TTT TTT ÖCS CCS TTG AGT ATT GAT GOT GAT M Ma Thr Thr Val 01 «Phe Phe Alá Pro Hév S®r n © Asp oly Asp Lys

755,720

GTO ACC SINCE ATT row ACT GGT ATC ÁCS GGS GCT dó CCA AAO AAC T00 With Thr With Ile Oly Thr Oly Ile Thr Oly below Leu Pro Ly® Asn Trp 705 770 775 % TTA OAO TAT GGT CAG THERE AAO CTA CAG OCA ACA ooo GGC AAT 80A AAA Leu Ola Tyr Oly Rail VSL Lys Leu Gin Ala Thr Oly Oly Asn Oly Lys 790 725 792 BROAD ACA TOO AAA TCC AST AAT ACT AAA ATT GOTHIC TCT OTT DAM AAG TCO Tyr Thr Trp Ly® Ser Ser Asn Thr Lys lle below Ser With asp Asn Ser ?> 5 200 SOS OOA CTG OVER THE ACC TTA AAT G & A AAA GGG AGT GCC AGA ATT AGT CTA SINCE Oly With XLE Thr Lee Asn Glu Lys Oly Ser below Thr XLE Thr With with 2X0 9x5 22.0 TCT row DAM AAT CAO AGT GCG ACA TAG ACA ATT aa! GCA CCG GGT AST Ser Gly asp Asn Rail Ser below Thr Tyr Thr Ile Asn below Ar® Oly Ser 225 230 835 240 ATT SINCE ATT THERE OTS DAM AAA AAT ACT CGA THERE ago TAT TTT DAM OGG Ile VSL Ile below With asp Ly® Asn Thr Arg With Thr Tyr Phe asp below 295 250 255 m AAC AAA TCT AAG ACA AAT AGG GCA AAT TTA GCA CAO CCA AAA OAA Glu Asn Lys Cys Lys Thr Asn Ser Alá Asn Len Ala Gin Pre Lys Gin 200 9 "S 870 SINCE TTG SCO AAT ATS TAT TCA. ACA TOG GGT GGT GCA AAT AAA TAT CCT Leu Leu below Dig" Ile Tyr Ser Thr Trp Yeah al | Asn Lys Tyr Pro 975 920 4 225 TAC TAT TCT SALT TCT AAA TCA TTG ACT GOT TGO AT ^ AAA CAA TCT TCT Tyr Tyr Ser by ser Lys ser Flax Thr Alá Trp lle Lys Gin Ser Ser 290 292 900 TCT OAA CAO TCA TCA GGT SINCE TCA AGG ACA TAT DAM TTG THERE ACG AAO ser Glu Rail Ser Ser Oly vei Ser Ser Thr Tyr Asp L® " With Thr Lys

m

9x5

920

4095

4143

4X91

4039

422?

3 S

4323

4431

4479

4527

AAC CAO TTG ATS AAT OTT OOA OA AAC AAT AAO AAT OCT TTT TCT OTT Asm Sin Len Ti® Dig Val Gly V®1 Asn Asn Lys Asn Ala Phe Ser val

925,520,935

4575

402?

GTCTCATTTO SOAATAAOTT TTTCGTSTAT CTTGTTTOAT AGAGACCTTG TTTACCATAT

CTACAACATC TOCACAATAA AAMCCCTCC OA & OAOOGGO MOAXAATAA

AGA8AACGGA ATMGOTS & T

AATCGCGTCT ATOAC & T & GC

ATtAAG & SCA ATTGWOAS '«wcm mwm

STCCATCGCA TTTATAA7GG á »ASO ACATCAT2CT

GA & dCT8AT GCCAGTTC & T

Meccowc GmTTCAöTA

GTATCCCÖAT AAGAGGAAGA

ACCGTATTCC GTXAAAATAT

G7WTAATTA CRAFTS

Λ tgattcgcaa csccmm

AAGTTTGTTT TCrnTAG

VHW3GGAA AGCAAAACGT

OWCTAGCC AGGWsAGAC

ATCASACAAT GGCAASGIGT

GATSACCTTT CC8OSST &

<· «Ϊ́ (; (/ ^γ:: '1 1'':(:) {(: ('<) ((( ^:: 1: 1:)):>'mmATTCA AOT3 & AÖATA · <$?

ATTGAWCCT TSTKMO emcecsss gömstwa

AT & TOAGWt AACATTtTTA

TGG7ÖAACTT ACATCAACTA

ATGG & ATATT CATAACTAA2

TCAAAACACC GCATCTGTiT attaöscaa wwrrm '

AOTOS & TT ACTAGTTCAT

CAAAGGCAGT GCAAGSAAAG

GC & ASAGCSG CTTACTCTGA

TGGGCTGCTS GAACAGCA7G

- 40 ATICG75TGG GCGGGAGATO

CXCTTTCAG AAT8CTACCA

TCTTCATCGT SCCGCTGATT

GOTMW? CTGAAATMT cogg & tctt ktctsotk

TTTTWmT & TTGMATA5 τοαττημτ Grmcwrm

ACASATTTIC AACTTCSCTC atccwot ^ wcm

CATTAATCGC GGiVSATOATT

WTCTTÖ © GTAATTMAA

GCGO3ACATT TrmX & TSC

GCTGGTA2 & C ATCAGCTCTÖ

MCTSCCQ & T GGCTCCAGAC

Gcrrrmns tcoatttttg

8S7CAATATC ACCAATGA7G

AT8 & OT & TC TSCTTCATAG

AMS8AAAAC ACCTOTCA wm »c sg®em

ATTSCCGATV ACÖC & TCOC &

AATTTTTTTC ATCCT38W3

ACAATAGAG? AGAAAGGAAG

GAT7 & CGTG & OTTCCAA'TG

TXAATSCTAA TTC & n & HER

CTMTAGTTA ttctccatea

CbSTTTCTGA AACGATTGAA

AASAG8AAGG WSACAACG

CCGATASCGA ΤΑΜ2ΤΜΑΑ

A2CCTAATTT © TCTATGITT * * »♦ ♦♦: * *» »* x *« * x · * ♦ * * »» 4 · »X.

'TOATACCAT CTTTAGGATC & TTCCC & 3GC TKJIACCG'TT l ((() {ij _: (((l <<l>: ((<l ( ^: Í))) {(1 (() {((() Ö <<< (( )) ((

C1UVERATED TTCCGGGSM l ^ O) l) l ^) l))) t) 8) ll8) l) ((((((8 (·)

MmsAmr TAcr & rrm

TGT2GMAAA CCnXCCCOC

TTTATATTAT TATTCrmr

TT & TGTMTT CCS & TCCAGC anrWTG AAÖGGGGAAT

GT & ATGATSA TATCCTTATA

WTWmC AAATGATATT

V *

AAW3AGTTA AATCAGTGGT γ **; · *

C&T & CSAGAö TGATTGCATG

CTAÁACGCTC GGGGrfCAGC

AAOGCAATCG CAGCACXAAT scmocc TTTCTGGCAC

AAGCAACAtC CCGCT & T & TC

Ί lx ·

GGGGTO3SCT CTCG & TCMT 'ol

CS7ATA2CAA CTAAAAACTG e

TOTXTGTGC CTAWGATAG

CCATTSAGAA όΤΓΠΑΤΓΠ ¹

-TWAAOATG Ά8ΑΑΑΑΑΑΤΑ

CTCTTGTACA FIVE TACTATS

8CM3S® TTGAATOTAA T ^ AATTGÖ MTCTOAACA TTAAATTTAC AAAA'TÖAATT G ^ CTAAGCC TGGGATATCG GAGA & 3CTGC TVAATGATAT

GAACTATCAT WUWAC & 8

G8TAA7A1GC CAAAAdGGGA «8?

4537

4 '?

5887

518?

5157

5227

52 '?

8347

5857 e?

852?

5587

5547

5787

5757

5537

58S7

588?

5087

8857

5127

5187

5247

5387

8357

8527

8457

5557 * **** »# ♦» Φ Φ * * * * * XX X ♦ »Χ« «χ

- 41 CATTGTTCCA CTTATATCTT CTTTATTASA ATCTAAGTTT GTTMCATT3 AATTAMAA OS »?

CAAATACCCC AAATTATTAT TAG & TTT & TT ASGCGMÖAT GATTCCCAGT TGGCTCTGCT SOS?

TTCCTGCTTA GGGGTSSSTG AGT7AAATCA GG & AGCTATC CAS & AG & TCA AAAAGCTTTA 072?

TGAAAASCCC AAGGATGAgG ATTCTGAAAA CCGAGCCTCT TTpJSACT CCTTTATGCA S787

CATTAAGCAT CTTCCAGAGC GTS & CA & CCA CTTAAAGGTT .J ^ «86 © CGGTATCGTT 0S47

CGATSTGTCT TATATGTCTT CTTTTGAAGA TAAGGTGMA ASATCTTCAA TTATTASCGA SS 07

TTTA7GCCGG GTAATCATTT THTATCGCT TGATAACTAT ACAGATATTA TTCCAATCTC 0307

TATT.AATAAA GATAAAGATG TCATTTTAAA TGA & GTATTG TCTATTATTG AACATGTATG 702?

GTTAACAGAA GACTGCCTCC TSG & AASCCC TTCTCGGGTA TCGATTGTCG AACATAAACA 7087

TGTTTATTAT TTCCACCTAT TG & AAG & CTT TTTTGCATCA TWXTCACG CTTGTTTTAT 7147

............. 0

TCATAGGCAA CAGAGAGAXA ATACATTATT AATGATTGGT AM37 »TO ATTATAACCA? 287 '· f' $ gg & tgttatt tgatattggt tttttaatcs ttttlggtct tgctaagaaa gattattaag? 3«? «. . * #

AGCTATATAC ATG GAT ACA. TCA ACT GCA ACA TCA GTT. GCT AGT GCG K 7310

Bet Dig Thr Ser Thr Alá Thr Ser Vei Alá Ser ΑΧ »Asn

IS 10

GCC W ACT TCG ACA TCG ACA CTG TAT GAC TTA GGC AOT ATG TCG MA 7304

Al® Ser Thr Ser Thr Ser Thr Val Tyr Asp Lee Gly Ser Bet Ser Lys

28 25

GAC GAA GTA GTA GTT CAG CTA TTT AAT AAA GTG GGT GTT TTT CAG GCT GCG 7412

Asp Glu Vei v®X Gla Lén The Asn Lys Vei Gly V & j ^ Ohu Gin Alá Alá _; JS 48 <5.? '· Ski:

CTT CTG ATG TTT GCC TAT ATG TAT CAG GCA CAA AG) GAT CTG TCG ATT 7400

Len Leu Met Phe Alá Tyr Bet Tyr Gin Alá Gin Seri Asp Len Ser Ile

SS - «0

GGA AAG TTT GCT GAT ATG AAT SAS GGA TCT AAG GAG TCA ACC ACA CCC 750 »

Ale Lys The Ale Asp Bet Asn GXe Als Ser Ly® Gla Ser Xhr Thr Al®

70 75

CAA AAA ATG GCT AAT CTT CTG GAT GCT AAA ATT GCT GAT GTT CAG AGT 755 «

Cin Ly® Bet Ale Asn Leu WX Asp Under Ly® Xlé Under Asp Val Gin Ser «8 85 88

AGT TCT CAC AAG AAT AAG AAA GCC AAA CTT CCT CA ^> CAA CTG ATT GAC 7004

Ser Ser Asp Lys Asn Lys Lys AX® Lys Leu Pro Glu Glu Val Ile Asp '100 XOj

TAT ATA AAT GAT CCT ± GC AAT CAC ATT ACA GTA GOT ATT AGC GAT 7053

Tyr XX® Aon Asp Pre Arg Asn Asp Ile Thr V & l Ser Gly Xle Ser Asp

210 1X3 228 125 # ♦♦♦ ** ♦ *

Κ 9 * ♦ <

Φ φ

ΦΦ Φ »» *

GTA ΑΑΤ Leu Asn CT? Als CTA Glu TTA Leu 13 8 CTG GG? GGT GAT TTG CTA AGG CTG AAC GCC GCT 7700 Gly Als Gly Asp Lea 135 Gla Thr VSL Lys Als Alá 140 ATT TCG GCC AAA TGG AAT AAC TTG AGG AGG SINCE AAT AAT AGG CAC 7748 lle Ser Als Lys 145 ser .. Asn Asn Flax Thr Thr 150 With with Asa Asa Ser Gla 155 CTT δ » OVER THE CAG GAIA ATS TCT AAT ACT TTA AAC C? A TTA AGG ACT GCA 7735 Leu Glu Ile 1 SO Gla Glahefc Ser Asa i «5 Thr Leu Asn Flax Flax 170 Thr Ser Alá CCT TCT CAT ATT CAC TGA CTG CTA TAG AGA ACT ATT TCA GGA ATA TGG 7844 Arg Ser 175 aap Ile Gla Ser Leu XSÖ tin Tyr Arg Thr Ile 185 Ser Under Ser OT GCT Leu Gly m TODAY Lys TAA CCGG & GAT & A < ET ATS OT AAT GTA AAT AGG GAT ATG Mer Leu Asa Vsi Aeu ser Asp Xle X, 5 w 7833 c & o tgt ATG AGG TGT CTG GGC AGG GGT CTT AGG GGT ACT TCA GGT ATT 7840 Gla. Ser Shoot ífet Arg Ser Gly Als 15 Ser Als Al® Thr below I & Thr Ser Gly 11® AAT CAG CCT GAA CTG ACT TCC GCT CTG GAT TTA CAA CTG GTT AAA TGG W Asn Gin IS Ere Glu Vsl Thr Ser 38 AX & Len Asp Len Gla 35 Flax Val Lys Ser 4o ÁS gcg CCA TCA CGA AC? TG? AGA CA & AC? AGG CTT TTA GGA AGG CTA $ 803 The Alá Tro Ser below "5 Ser Trp Thr Glu Ser 50 Thr Als Leu Fre Thr Pre 55 GCG CGA GGC CAC TGA TTA CTG ACG CCT TCT GTT GCT SECURITY CAT CTG CTG 8ÖS4 Fra Als Gly His Salt Ser Leu Vsl Thr Fro Ser SS VSL below Gin ASp Vsl Leu 70 BRANCH? AA & CTG TTT GG? GGT ATT AST CCT CAA CTT ACT AST CGA AGENCY 8137 Ser Lys Leu 75 Phe Gly Gly lle Ser 88 Gly Gin With Ser SS Arg Thr Gin CTA AGG GA & GCA CAG CGC AGG ACT CAG AAT GCA AGG TCT GCG TAT GCT 8188 Gly Thr Fö Glu Fre Gla Arg Ser Thr $ 5 Gla Asa Als Ser 188 Ser Sly Tyr Pro TAT OT TGT CAG CTG AAT AAC GTT CAG CCT CAG CTG ATG ATG ATG ATC 8320 Tyr Len X05 Ser Gin VSL & sa Asn 118 vei Asp Fre Gla 115 Als Met Met Week Week 110 CTG AGG CTG TTA TGG CTG GAT CTT TGA GCG CAG Ahh CT? GGG ACT ATC 827 ' Val Thr Leu Leu Ser 13 5 Leu Asp below Ser Ala 138 Gla Lys <$ *. with Down Ser Week 13 S AAA AAC ÓMjdAudrik TGC AAC GAC ATG TAG ACT CA? CCT CAA AAT AAG GCG CTG CAT 8324 Lys Aaa Ser Asa Wingfield Glu lle Tyr Thr Asp Oly 145 Gla Asa Lys Below Len Asp 150

«9 ΦΦΦ Κ + φφ * φ

AAT Price AAA Lys AGG Thr 155 CTG Leu GAG Gla TTT Phe AAA Lys AAG Lys 15 « CAG Gla CTG Leu GAA Gla GAA Gla CAA Gin X55 CAG Gla AAA Lys GCC Ale 8372 GAA GAG AAA GGA CAA AAA AGT AAA ATT GTT GGT CAG GTC TTT GGT TGG 8420 Glu Gla Lys Ale Gin Lys Ser HYS Ile Lee Gly Gin vei Phe Gly Trp 17 « 175 18 « TTG GTC GCG GTA. ACT GCT ATT GGA GCT ATT TTT ' AAC CCG GCA CTG 3455 Pee Gly vei Ale vei Thr Ale XLE Ale Als XLE Phe, AEE pre below Leu 155 130 135 20 « TGG GCT GTT GTT GCC GTT ACC GGA AGA GCA ATG GCA CTG CAA AGT GCA 8515 Trp below with vei Ale vei Ser below Thr Ale Met Ale Leu Gla Thr below SOS 318 315 GTT DAM GTA ATG GGC DAM AAG GCC CCG GAG GGG TTA AAG ACA GCA GCG 8554 vei AEP m mse Gly aeg Lys Ale «from gl " below Lee Lys Thr below Ale SSG 335 33 « GAA GTG TTT GGC GGA GTA TCT ATT GCT GCA AGT ATT :, CTG ACA GCC GGC S512 Gla 1% The Gly Gly Val Mud He Ale Ale Ser XLE Leu Thr Ale Gly SIS < sec .345 ATT GGC GGG GTG TCT TCA GTT ATG Φ »Φν> JL. AAA GTT GGC DAM GTC GGT AAC 055 ' XLE Gly Gly vei Ser Ser Lea hefc Ser Lys vei Gly asp vei Ale Asn 250 355 350 AAA GTT GGT TGA AAC ATC GTG AAA AGA GTT ACT AAA CTG GCC DAM GTA G7 "8 Lys vei Gly Ser AEA Ile With Lys Thr vei Thr Lys Lee Ale AEP With 255 370 375 30 « TTT GTT GAG AAC GTT GTT TCA AAA GTT GCG GCA ACG GCA AAT GGA TTT 8755 «he vei Gla Asn vei With Ser Lys vei below Ale Thr Ale aaa Gly «he 255 28 « 305 ACG ACC TCT GCA CGT TCT ATT GGG ACA ACT GTG CTG AAT AAT DAM TCT 8004 Thr Thr Ser below Arg Ser Ile Gly Thr Thr With Lee Asn AEA asp Ser 30 « 7 305 31 « GCG GAA TAT AGG OVER THE TTG GCT CAG TTA TCT GCT TAC GCG GTA CA & AAC 0852 Ale Gla Tyr Ser Ile Lee Ale Gla Leu Ser Ale Tyr below With Gin Asn 315 330 335 TTA ACT GGA CAG AGT GAA AAC CTG GGT GAG AGT GGG AAG GTT GAG CTG 8300 AEE Thr Arg Gla Ser Glu Asn Leu Gly Gla Ser below Lys VSL Gla Leu SS® 335 w DAM AAA GCT GCA GCC GAG TTG GGA AAC CAG GCA AGG TAT TTA CAA AAC 8540 asp Lye Ale Ale below Gla leu Arg AEA Gla below Ser Tyr Leu Gla AEA

Ser

GCT TCT CAG ΤΤΑ ΑΤΑ TGG GAC TCA GCA CGA GXA & ΑΤ AGG CGT ATT GTT Ala Ser Gla Leu Xle Ser Asp Ser Ale Arg Val Aaa Ser Arg lle Vei 378

355 * * ♦ »# ♦ ** ♦

Λ *> * * ♦ * * * * * ♦ # »« · * ♦

AGT © GT CGA © η TAA * TTATTAAAGA GAATTTAATT ATG AAT ACT ATT GAT Two Asa Thr Xle Asp 5045 Ser Gly Arg Wei 350 1 5 TAT ACT AAT CAA © TA AT © AGG GTT AAT TCT GTT TCG GAG AAT ACT ACC never Tyr Thr Asn © le vei Met Thr va l Asa Ser With Ser © lu tos Thr Thr 10 X5 20 GGC TCT AAT GCA ATT ACC GCA TCT © CT ATT AAT TCA TCT © TT CTT ACC 3X42 © ly Ser Aon Ala XLE Thr Alá Ser AX & XLE Asn © er © er Leu Leu Thr 25 30 35 DAM GGT AAG GTC DAM GTT TCT AAA CTG ATG CTG © AA ATT CAA AAA CTC 0150 asp GXy Lys With asp VAX Ser Lys Lee Mer Leu Giu Xle Gin Lys Leu 40 45 50 CTG GGC AAG ATG © X © CGT ATA TT © CAG DAM © TA CAA. © CA CAA © CA © TT 5230 Leu ciy Lys Met With Ar® xle Leu © la top Tyr © In Glu © la Gin Leu 55 SALT SS © TG CAG AGG TAT CAG ATC CAA CTG GCC GTT TTT © A © AGC CAG AAT AAA 3280 Ser Gin Ser Tyr © la, Xle Gin Leu Alá With PH ® Glu. Ser © la tor Lys LAKE 75 00 S5 GCC ATT DAM GAA AAA AAG GCC © CT © CA ACA GCC GCT CT © GTT © GT GGG 3334 below XLE asp Glu Lys Lys AXa Ala below Thr Alá Down Leu With © ly Gly 00 SS 100 GCT ATT TCA TCA GTA TTG GGG ATC TTA GGC TCT TTT GCA GCA ATT AAC 3302 Below. XLE Ser Ser With Lee. Giy Xle Leu Gly Ser Phe Al® Aia XLE Asn 205 110 115 AGT GCT © AC AAA GGC © CG AGT © AT ATT GCT CAA AAA ACC © cc TCT ACA 3430 Ser below Thr Lys Gly Today Ser Asp XLE below © off Lya Thr below Ser Thr ISO 125 . 130  7 * '· TCT TCT AAG GCT ATT DAM GC © © CT TCT © AT ACT GCG ACT AAA CARPENTER TTG 5470 Ser Ser Lys aXa XLE asp Down Aia Ser top Thr Alá Thr Lys Thr Leu 135 140 245 ACT AAG GCA CARPENTER GAA AÖC © TT GCT © AT GCT © TT GAA GAT © CA TCC AGC 3525 Thr Lys below Thr Glu Ser Val Al® asp below vai © lu Asp below Ser Ser ISO 155 150 XS5 © GT ATG © CA CAA GC © ATG ACT ACA © CA AC © AGA © CG GCC AGC CGT ACA 3574 With Met © In © In Alá Thr Thr Aia Thr Ar® Ala Ala Ser Ar® Thr 170 175 4 ' «· XOO TCC GAC GTT GCT DAM GAC ATT GCC DAM TCT © CT CA © AGA GCT TCT © CA SS22 Ser top With below top asp Xle Alá top Ser below Gin Arg Ma Ser © la 105 ISO 105 © CT GCT GAA AAC © CT GCA GAT GCC © CT CAG © AA © CA AGT CGG © CA AGC © 3S7 Lee below Putty Sh & Alá Alá Aep Alá below © la Lys AXa Ser Ar® Today Ser

200 205 2ΧΟ

Φ 4 *,. φ W * ♦ ♦ Φ * 9 9 Μ

X φ Φ * Χ · φ * * * · φ φ

Χ. «Φ 4 * Χ« V · ΦΦ * Χ

O3C TXT ATG GCT GCA CTA GAT AAG ATT ACT GGC TCT ACA CCA TTT ATT 8? 28

Arg Phe hht AXa Als Val Asp Lys He Thr Gly Ser Thr Pro Phe Xle

21S 220 225

GCC CTT ACC AGT CTT GCC GAA © SC ATS AAG ACA TTG CCA AGA. ACG GTA 975S

AX & Val Thr Ser Leu Alá Glu Gly Thr Lys Thr to Pro Thr Thr VaX

339 235 240 245

TCT GAA TCA GTC AAA TGT AAC CAT GAG ATT AGC GAA CAG CGT TAT AAG 38X4

S®r 81 «Ser Val Lys Ser Asn His Glu XX® ser Glu Glu Arg Tyr Lys

25 «255 280

TCT GTG GAG AAC TTC CAG C&S GGT AAT TTG GAT CTG TAT AAG CAA GAA 3862

Ser Val Glu Asn Phe Gin Gin Gly Asn X »e« Asp Len Tyr Lys Gin Glu

265,279,275

GTT CGC AGA GCG CAG GAT GAT ATC GCT AGC CGT CTG CGT GAT ATG ACA 551 ©

Val Arg Arg Alá Gin Asp Asp 11® Alá Ser Arg heh Arg Asp Week Thr

AGA GCC GCT CGC GAT CTC ACT GAT CTT CAG AAT CÖt ATG GGT CAA TCG 835S

Thr Al® Alá Arg Asp To Thr Asp Leu Gin Asn Arg Giet Gly Gin Ser

255 30 «505

GTT CGC TTA GCT GGG TAA TTGATCATGG TCGATACGTT TAATGATGAA 10995

Val Arg Lau Alá Gly *

21 «315

GTGCTTAATC ACTATCTTGA ACAAAAAGGG TACACA & T & C AGAAGGAGTT TCTTTGTGGC 10966

A8T8GCTTTT TTATCGGATG GCGGATTGAG ACCTCTTTTT TCTCATTAGC GTACAGACTT 10125 6 © ATGAACAAG AACTGATTTT GTGCTCTTTT GAAGCACGTA ACÖU & C & GCTTAACÖGC GG 10136 CCTGTTTTAT CACTGACTCG CTTGCTCGAA GAGTTGTACC C ^ TTTTTC GGGTATTAAG 18245 AAAATCAGTG CGATGAAATŐ TAAGATTGST TCAGATTCA © AACGTCAAAA GCGCGAAGAG 10385

TTGTTTAATT ACTTCATCAG AAAGGGCGCT 62GCASCAA6 AAACAGAAGA CGGAATTTGG 18355

TTCGTAAVGA ATGTAAATAG TTACAAATTT TTATTTCTAT TAACTATTGA GGAAAATTTA 10525

ATGAATTTAT CTGAAATTAC TChACAAATG GGTGAAGTAG GTAAAACGCT GAGTGATTCT 195 8 5

TTTCTGCCAA CAAGCTACTG GTGTGGGCGG CTGTAGCTGC GGCAAATCAT AAGCTTCCCA 10726

AATAT8C & GA ATCTATCCTG GATGTATXGC CGCAAATTAT ACCCGATA & A AAAGATATCG 1078®

CACATTTAGA ATTTATTATT TTATATGGAT TAAATAGAAA AAATOATGCG GTAAASGCTC 10845

TGGAGGACTT TATGGATGAT GAGACAAGCC AGTTGTTATG CTGTCTGGTC CACGAGAATA 18506

ATAGAAGCTS GACACTAATG

GTAC0AAA2T TTCTMGTTr

TTWAK ATHLETIC

TATGtTTAM GGAATTAGTC

AAÖCCGÖQTA ASCTCWTOG

GAATSCSAXT AÖW2TCAAT

GACTTTTMT osactagcc

GTOSACAAGT SGAOÚ3XCTT

AAAAGATCAT TTAGCTSCCT

AAGCCGCCCS GCACCGGGGS

ACXGCCCACT GCTCM ^ CÓC

WATACTSA SGTCTTAAOC & TCCGASGC & TCCSSS ^ CÁT otatgaactg tcsaaaaagg

TTTAGASTA · TA&&& TAKE IT

ATATCWXT2 «M2GAAAX

AÖW3GÖT3T XTCSTTAACC

AXGTTSGATl 'TGACATATCA cstatttttt ttetwra®

Aöcxsmtím WSTGGG3S.

AWCTTTTGG AAGAACTGÖT

TACTrCTCXA CTCACTfóXT

ÖATTATGASA TGGXTS & TAG

CTTTTCAATX ®ΤΠ »Μ

AGCACAATAT TTGTCAATTC

TCTOTCCAG 'SCATTAAIAT

QTTCAGCGXA TTTCTSÖ & TA

ATCAGATTTC STCÜAGCAGG

- 46 ~ ttttaatctc atattgagtt

TAACAGAGAT ATTTATAATA cawww? nsw? w wwtssts romw tóswmc 1ACAC1AGGA

OGTTATCTGÜ CTTTTCACTG

CTCOTTTTt TCCCSGXAC2

OTgeSASTTC ATTTACTOSG ccsssccatc coAxcrm

AXSAAAAXXC SAAAAGTSTT

ÖGCCGACAAG CSGGTTGGTA

TAAAAGAXCA TTTGGCXGCC

CAAGCCGttt GGCACO3CCG

TACC8CSTAT TGCTCAÖ2CA

C & TAAAAAGT ΑΟΑΤΓΓΑΑΑΑ

AAACWATAT T && STTGCAT

AAAXAXTG1A ATMAXGATE WORK

ASAffiXXÚTA ATTTTTIATC acacia ssssome

Í5T »» TAICTCIATT txxcatcwx TAíseteros

CTGOTWTG ATTGÖCTATT

T8CTAASC & T ATTTATTTTA

AGCTTTÖCGC XGGXATGAXA

CTCTTTGGCG XAOTCACTO

CCTWrWAT CACGCGAOAT ctthsgt & ct rmrceoGCG

ACTOATAGT OTCTWCC

ACTGTTTCTÖ ÖATA & ASCTT φ $.> · ...> <** »X ** ♦ * * * * XX 0 * * *» x * * * 0 * · * * # * 0 -0 «« * i * * « * »

TAAACMTTG ATAMtTTCA

CCTTMTGAA AAAXAX ^ GCT

AGTTTATA3T CGtTAACTAT

ASATATAAAA AGGSATGA & T

CGÓCATA11A CTAÜTGCGGC

WCCTCWG CTSTTCGTCT

TOMATATSA ATGCGAO & AC

TCTCGTCtW CCCCACCGCC

COXCCCATTG CTCAGGCATT

ÖATACTTCAA ÖSCTTAAGCC

TCÍGGGTCAT GCCGGTCTTX

TATGAAAA & X CGAAAAGTCT C ^ CCCACAA GTGGACAOGC

Λ \ 'γ

TTAAAASATC AWWCTSC

TTATTAATTA CTGTTÜATTG

TCTGATAAAT GTSATTGGTA

AAAGAGWTA ATAGAGCAAG

CA1AAATTTA CCGC1ATXTG

TGX & TGTACA GGC1CTTATA · * <· k.

XAWCCGATA 2dASGCT3GC Av

AihoAC & T ertmcTAAT ACATCCACTG TCTGGTTCTT

TCACOSAA & C SACCATTXC

TTCAACTXGA GACTGXAATG

GAAATGTTTC TGOTATTTTC

GWTAA & CTO XTCGCXCATC

ACGGAGGTCA AAATTCOGTT

AWJAACATC GCW3AT & TC • '\ · ν:.

xo $ s "

1X22 "

XXÖSS mo

XX2ÖS

1120

XX32S

ΧΧ3Ο

NEW nsM

XX5O

11S2S nos xxw xxs © «

XX8GS »538

XX98S

X284S

1210 «mss

12228

1228 «

12x4 "

X24®8

124 ««

1252 «

X2S8S

12 «2« * ♦ «# ♦♦ * *» y * ♦ «κ ♦» * * * κι * # 9 «<Χ4Γ ί» Λ »<· Χ

- 47 SEQ ID NO: 2 SEQUENCE FEATURES:

LENGTH: 940 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULATE IPVS: protein e

DESCRIPTION OF SEQ ID NO: 2:

Met Ile Thr Hi® Gly Phe Tyr AI® Arg Thr Arg His Lys Hi® Lys Le » 1 5 Shoot IS Lys Lys Thr Ph © Ile Met Leu 2® y Ser Ale Gly Leu 25 Gly Leu Phe Phe Tyr J 3® With Asn Gin Asn SeriPhe Alá 3S A _: g < Asn Gly Glu Asn 4® Tyij Phe Lys Leu Ser L · · 45 Ser Asp ser Lys Lee Lee Thr Gin Áss Alá Ale SS SS Gin Asp Arg Leu Phe 5® Tyr SS Thr Leu Ly® Thr Gly Glu 7® Thr Val Al® Asn 75 Ti® Ser Lys Ser Gin SS Gly Xle Ser Leu Ser Vei He SS Trp Ser Leu Áss. 0® Ly® Hl® Leu Tyr Ser 55 Ser Glu Ser Glu Week Met Lys 10 ® Ale Gly Pro Gly 305 Gin .. Sin ile Ile Leu Ú 110 Pro Leu Lys Lys Leu .Ser Vei I1S i Glu Tyr Ser Aia 32® LeuPro Vei Leu Gly Á 125 Ser Al® Pro Val Val Aia Al® 136,325 Gly Gly v »X Alá Gly Mi® Thr Asn Lys 14® m 14S Thr Lys Seven Ser Pro Asp 1 SO Ale Thr Lys Ser 155 Asn Thr Thr Asp Asp IS® Lys Ale Leu Asn Tyr Al »Al® 3SS Gl »Gin Al® Al® 17S Ser Leu Gly Ser Sin 175 Leu Gl »Ser Arg Ser Le» Asn is® Gly Asp Tyr Ale 1S5 Ly © Asp Thr AI® Leu IS® Oly week Al® Ser Ser Gin Ale 1S5 Ser ser Gin Leu 200 Glh Alá Trp Leu Gin 205 ca. Tyr Gly Thr Al® Glu Val 21® '235 Asn Leu Gin Ser Gly Asn Asn Phe Asp 22® sxy 225 Ser Ser Leu Asp Phe Leu 250 Leu Pro Phe Tyr 235 Asp Ser Glu A »» Met 24® Leu Ale Phe Gly Gin Vei Gly Ale Arg Tyr ile Asp Ser Arg Phe Thr

24S 25® 255

X fc «4 ♦ fc · ♦ ♦ ♦ 4 ♦ ♦ 4

Aln Asn leu Gly Alá Ile Gin Arg Phe Phe Phe Le® Pro Gin Asn 278 Asn Ke® Thr Flax Arg Gly Tyr Asn 258 Asp Gin 288 255 AEP 8er Gly Asp $ 23 With Phe 275 les Gly Ile 288 Gly ^aly Gly Tyr Trp Arg Asp Tyr 285 Phe Lys 388 Ser Ser vei Asn 385 Gly Tyr Phe Arg heh 318 Ser Gly Trp His Gin 315 Ser Tyr Asn Lys Lys 320 asp Tyr Asp Gin Arg 325 Pro Als Asn Gly Phe 330 asp ile Arg Phe Asn 335 Gly Tyr Les Pm Ser 348 Tyr Pro Down Lee GXy $ 34 Als Lys Lei Two Tyr 388 Gin Gin Tyr Tyr Gly $ 35 asp Aen With Under Len 358 Phe Asn Ser ASp Lys 355 Flax Gin Ser Asn Pro Gly $ 78 Ale below Thr Val Gly 375 vei Asn Tyr Thr Pro 388 Ile Pro Flax vei $ 38 Thr Get «X Ile asp 388 Tyr Arg Als Gly Thr 385 Gly Asn Gin Asn asp 488 Flax Len Tyr $ ®r Kei 485 Gin Phe Arg Tyr Gin 418 Phe Asp Lys pm Trp «15 Ser Gin Gin Ile Gin 428 Pro Gin Tyr hour Asn 525 Gin Flax 3A Arg Thr Les «38 Ser Gly Ser Arg Tyr 835 asp Flax With Gin Arg «48 Asn Asn Asn Ile Ile «45 Flax 81® Tyr lys Lys Gin 558 asp Ile Flax Per Leu «55 Asn Ile Pro Small Asp 558 Ile Asn Gly Thr 555 Gin Ski Ser Thr Gin 478 Lys lie Gin Flax lie «75 Bye Lys Ser Lys Tyr 4S0 Gly lsen Asp Arg Ile With Trp Asp Asp Ser Als Les Arg Ser Rail Gly

$ 48 «80« 85

GXy Sin Ile olla Small 8ly Oly Ser Sin Ser Us Gla Asp Tyr Gin Alá w salty sic

Ile Leu Pro Als Tyr Val Gin Oly Gly Ser As® Ile Tyr Lys Re Thr SIS §2 & 52S

Als Arg Als Tyr Asp Arg Dig Gly Dig Ser Ser Dig Dig & Gin Le® $ 538 35 S «0

Thr Ile Thr Vei Len Pro Dig Gly Gin Vei Asp Gin val Gly Val 54 $ 558 SS5 SSG φ φ

Thr Asp Phe Thr Alá Asp Lys Thr Ser Alá lys Alá Asp Gly Xle 875 Putty 555 570 Miss Xle Thr Tyr Thr Alá Thr Val Lyg 589 585 lys Dig Gly Val Alá Gl® 559 below As®val Pr © val Thr Phe Ser Xle Val 985,890 Ser Gly Thr Alá 895 Thr juice Gly Under As® Ser Under. Arg Thr Asp Oly & s & Sly lys Alá Thr 810 815 820 Val Thr became Lys Ser Alá Thr Pro Gly Sin Val Val 825 810 Val Ser Als lys 855 Thr Alá Putty 840 Met Thr Ser Pro leu A® »Alá Ser Ale 885 val xle Phe Val 880 Asp Gitt 855 Thr lys Alá Ser Xle Thr Gle Xle lys Alá 889 885 Asp lys Thr Thr Below Lys 870 Al® Asp Gly Ser Asp Alá Xle Thr Tyr Thr 875 859 Val Arg Val 'Mer 885 Lys Gitt Gly Under Pr Val Val Asp Gin lys Val Thr 889 SS5 Phe Ser ly® Asp 799 Phe Gly Thr lev As® lys Thr Gle Alá Thr Thr Asp 795 719 Gitt Áss Gly Tyr 715 Al Thr With 720 lye les Ser Ser As® Thr Pre Gly lys 725 Alá Xle Val Ser 730 State 735 With Ser Oly Val Gly Thr Gitt Under 740 745 Thr Thr Val Gitt Phe Phe 750 below Pre Leu Ser Xle Asp Gly Asp lye Vei 75S 780 Thr Val Xle Gly 788 Thr Gly xla Thr Gly Provided Pro lys Asn Trp le® 770 775 01® Tyr Gl / Gitt 750 val Lys became Glh Alá Thr Gly Gly Asn Gly lye Tyr 785,799 Thr Trp lys Ser 795 Ser As® Thr 990 lys lle Alá Ser val Asp As® Ser Gly 595 Val Xle Thr le »910 As® Glu §15 Lys Gly Ser Al® Thr lle Thr Val Val Ser 820,825 Gly Asp As® Gl® Ser Alá 950 Thr Tyr Thr lle As® Alá Pre Gly Ser Xle 535 989 Val Xle Alá 585 Asp Lys AStt Thr Arg Val Thr Tyr Phe Asp Alá Gitt as® Lys Gye Lys Thr As® Ser

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SEQ ID NO: 3 SEQUENCE DATA: SEQUENCE FEATURES:

Hero S E A: 193 am no s a v

TYPE A: acetic acid

TOPOLOGY: 1 i n-eári s.> Οώ

DESCRIPTION OF EXVNCIA:

Not" asp Thr Ser Thr below Thr Ser With below Ser aXa Asn below Ser Thr X 5 20 25 Ser Thr Ser Thr With Tyr asp juice" Oly Ser net Ser Lys asp 82 » vei 20 25 30 vax 81 » Juice" Phe Dig" Lys with 8Iy With Phe Gin below below Juice" Lee net 35 20 <5 Phe below Tyr Met Tyr 81 » below 81 » Ser asp LStt Ser XXe below Lys Phe 50 55 80 Als asp net Dig" 81 ») below Ser Lys 81 » Ser Thr Thr below 01 » Lys net 55 'TO 75 80 Α1 » Asn Leu With asp below Lys Ile below asp VAX Rail Ser Ser Ser asp SS 30 35 Lys Dig" Lys Lys aXa Lys La » Pro 81 » 81 » With He asp Tyr 22 « Asn 200 105 120 asp Pro Arg DIG" asp XXe Thr vei Ser Oly XLE Ser asp Juice" Asn below 115 120 225 Ski". Juice" Oly below Gly asp Leu 81 » Thr With Lys below below Ile Ser below 120 135 240 Lys Ser Asm Dig" Juice" Thr Thr With With Un Dig" ser 81 » Juice" 81 » Ile 245 ISO 155 ISO 8ln 81 » Met Ser DIG" Thr Juice" Dig" Juice" Juice" Thr Ser below Arg Ser asp 1S5 170 175

y »# 6 9« · * * '»« ♦ Λ »ν *» »* ♦ *

Φ «Κ χ * ·» * * ♦. ♦ ** «* ♦ ·« e

Ue din Ser Leu Sin Tyr fcg Thr Ih Ser Alá Xle Ser Leu Gly Lys. _Λ t * c 190

IDENTIFICATION 4 IDENTIFIC SEQUENCE DATA: SEQUENCE CHARACTERISTICS:

LENGTH: 381 amino acids

TYPE: amino acid

TOPOLOGY: linear

4. IDENTIFYING IDENTIFIED SEQUENCE DESCRIPTION: Mer Le »Asn Val Aaa Ser Asp ile Gin Ser Met Ar Ser Gly Alá Ser 1 5 18 15 Alá Alá Thr Alá Thr ser Gly Ile Asn Gin pr © Glu val Thr Ser Alá 28 $ 2 38 Len Asp Len Sin Len val 5yg Ser Thr Alá Pr © Ser Alá Ser Trp Thr IS '48 45 Sin Ser Thr Alá leu Pr © Thr Pr © Pro Alá Gly Little Ser Leu with Thr 58 55 88 Pro Ser Vei Alá Sin Asp Val Len Ser Lys Leu Phe Gly Gly Xle Ser SS 70 75 88 Gly Glu Val Arg Ser Arg Thr Sin Gly Thr Glu Pr © Gin Arg Ser Thr 85 28 25 Gin Asn Alá Ser Ser Gly Tyr Pr © Tyr Leu Ser Gin Val Asn Asn Val 188 ν 18s 118 Asp Pr © Gin Alá Mer Two Weeks Met Val Thr Leu Leu Ser Leu Asp Alá US 138 185 Ser Ale Gin Lys Vei Al Ser Ser Lys Asn Ser Asn Glu xle Tyr Thr 138 135 148 Asp Gly din Asn Lys Al Len Asp Asn Lys Thr Leu Glu Phe Lys Lys 145,158 155 158 din Leu Glu Glu Gin Gin Ly® Alá Glu Glu Lys Alá Glu Lys Mud Lys 185 178 175 Ile Leu Gly Gin Val Phe Gly Trp Leu Gly Val Ali Val Thr Alá Xle 188 1S5 188 Alá Alá Xle Phe Asn Pr © Al »Leu Trp Alá Val Val Alá Val Ser Alá 155> 288 285

ΧΦ * Φ ** fc fc «« ♦ * φ «'» ** $ ♦ * fc * S * «44 ♦« ♦ · * fc «» νΦ ~ 52 -

Thr Alá mt Π3 AiA Les 81a Thr Al »Val Asp Val h» fe Cly A »g Lya below 215 223 81 »Below le »lys Thr Als A) a 81 »rrl Ph» Gly 8ly Vei Ser XI® 225 233 235 2 »8 Al Ma Ma Ser lls Lea Thr below Giy lle 8ly 81y val Ser Ser ae " dig 258; 255 in hyg Val Giy Asp V & i Als Aa »lys Val Sly 8sh As» Xle With lys 288 2® »s? E Thr val Thr lys les Als asp Val Phe Val 81 »hsa Vei Val Ser lys $ 27 2S8 285 VAX Down Under Thr Alá Asm 8Xy Ahe Thr thr S »r Alá Arg Ser Xl < 81 y 2 SS 2 SS 388 Thr Thr Val 1® «Dig Dig" asp Mud Als 81 »Tyr Ser 11« 1 «» Als 81 » 389 319 313 328 Xísö Ssr Alá Tyr Als With 81 » As »t-eu Thr Arg 81a Ser 81» Dig" leu 325 338 A 335 Gly 81 »Mud Als lys With 81 » le »Asp lys Als Als Alá 81» Lse Arg 3 «.» 345,358 AS »8ls Als § »r Tyr Ims 81 » & »» Als Ser 81a les Xle Ser asp Ser 355 3AV 355 Als Arg Hours Asn Ser Arg 11 « Val Ser Cly Arg Val * 37S 37 » 388

ΑΖ 5 SEQ ID NO: SEQUENCE CHARACTERISTICS:

LENGTH: 315 amino acids

TYPE: amino acid

TOP OF THEOLOGY: 1 i n mud

MOLECULAR TYPE: protein

DESCRIPTION OF SEQ ID NO: 5:

WV AEA Thr 11 » asp Tyr Thr Dig" 81 » With Seven Thr Wei Dig" 8er With 1 5 18 18 Ser 81 » Dig" Thr Thr 8LY Ser Dig" Als XLE Thr Al »Ser below XLE THE"" ah 25 78 Ser Grayling ΙΑ » hee Thr asp Sly Lys x & y asp With Ser lys Les Fief Χλ " 15 48 . 88 81 « XLE 81 » Lys les X> »« 8iy hy » M »fc With Arg tle les 81 » asp Tyr 58 55 88 81 » 81 » 81 » 81 » A® » S »r 81a Ser Tyr 81 » XLE 81 »A» » below With Phe 55 73 75 58

* »« Φ ν * Φ * χ t * Φ 0 ϊ. Λ * * * * «» Χ «« »· *« * Φ Ή ν «

Gitt Ser Gla A® »Lys Al® XI» Aap Glu lya Lys Al® Alá Alá Thr Al® SS 90 55 Under Les Vei Gly Gly-Alá Xle Ser Ser 100 hangs With Les Gly Xle Leu Gly Ser 110 Phe Ma Al® 1X5 Xle Asn Ser Alá Thr Lys GXy Al® Ser Asp Xle Al® Gle 128 125 lys Thr Al® 130 Ser Thr Ser Ser lys AX® 135 XLE Asp Alá Alá Ser Asp Thr xao Al Thr lys 145 Thr lett Thr Lys AX® Thr 158 Putty Ser V®X Ale Asp Below 155 ISO Gitt Asp AX® Ser Ser Vei Kel Gla Gin 155 Al® 170 Met Thr Thr Alá Thr Arg 175 Alá Al® Ser Arg Thr. Ser Asp Val Ale ISO 18S asp Asp Xlé 'Alá Asp Ser Als 190 Gin Arg Al® X95 Ser Gin Lee Al® Glu Asa 290 below Al® Μρ Alá Alá Gitt lys 205 Alá Ser Arg 2X0 Alá Ser Arg Phe Two AXa 215 & X & Val Asp ly® Xle Thr Gly 220 Ser Thr Pre 225 Phe Xle Alá Val Thr Ser 230 Les Ale Gle GXy Thr Ly® Thr 235 250 Les Pre Thr Thr Vsl Ser Gla Ser Val 245 lys 250 Ser Aaa His Gls lle Ser 285 Gle Gle Arg Tests Ser Val Glu Aen 280 2 SS Phe Gitt Gitt Gly AStt Lett Asp 270 Les Tyr lys 275 Gin Gis Vei Arg Arg .Al® 280 Gla Aap Asp Xle Al® Ser Arg 285 les Arg Asp 2S0 Arg Met Gly Request Thr Thr Alá Alá Arg Asp 2 SS Sin Ser Val Arg lev Alá Gly Les Thr Asp les Gla Asn 300 4

305 '3X8 3X5

Claims (9)

PATENT CLAIMS 1) A method for producing a non-pathogenic E. coli mutant in a cytopathogenic pathogen in epithelial HeLa cells. B. coli mutant, which cytopathogenic effect is manifested in part by the fact that polymerisated .actin is formed by cables # that extend from one end of the cells to the other and that by increasing the amount of vinvulin - # characterized by the pathogen S, coli strain was mutagenized and mutants unable to induce cytopathic effect were selected. 2} A non-pathogenic mutant strain of E. coli characterized in that it was produced by the method of claim I. The non-pathogenic mutant strain of E. coli according to claim 2, characterized in that it carries at least one mutation that inactivates at least one gene of the LEE locus. 4) The non-pathogenic mutant B. coli strain according to claim 2 or 3, characterized in that it is derived from an E. coli strain pathogenic to rabbits belonging to the serum group 01.03. 5) The mutant strain according to claim 4, characterized in that it is pathogenic to E. coli strains deposited with the CNCM (French National Collection of Microorganism Cultures) on January 15, 1997, under numbers 1-1807, It is produced from the strain BIO. δ) Referring to FIGS. Non-pathogen according to any one of claims 1 to 3. mutant E. coli strain # characterized in that at least one ** · *. * <** »**«. »*« «9 *» ♦ »χ» carries a mutation resulting in the inactivation of at least one aep gene in the LSE locus. 7) Non-pathogenic mutant B1 / C & l .5. coli strain, deposited at the CNCM (French National Collection of Cultures of Microorganisms), January 15, 1997, under number 1-1808. S) A vaccine which is at least one non-pathogenic mutant of any one of claims 2-7. coli strain. 9) The sserint vaccine of claim 8, which is for oral administration, 10) In Figs. A mutant of any one of claims 1 to 4. coli strain for the preparation of a vaccine. 11) Use according to claim 10, wherein the vaccine is intended for administration to a rabbit.